0J.-9.
REMOVAL OF URANIUM AND MOLYBDENUM FROM
URANIUM MINE WASTEWATERS BY ALGAE
by
Katherine T. Dreher
Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Chemistry
New Mexico Institute of Mining and Technology Socorro, New M1exico lo)'3
9308180134 93080511 December, 1980 PDR WASTE WM-11 PDR TABLE OF CONTENTS
Page
Acknowledgements ...... ii
Abstract ...... iii List of Tables ...... v List of Figures ...... vi
I. INTRODUCTION ...... 1 A. Bioaccumulation of Heavy Metals ...... 1 B. Mechanisms of Uptake of Heavy Metals ...... 3 C. Algae, Metal and Sediment Interactions ...... 7 D. Objectives of Present Study ...... 9
II. MATERIALS AND METHODS ...... 10 A. Site Description ...... 10 B. Collection Procedure ...... 14 C. Laboratory Culture ...... 14 D. Algae Digestions ...... 14 E. Uranium Toxicity Test ...... 17 F. 24-Hour Uptake of Molybdenum and Uranium...... S...... 18 G. Long-Term Studies ...... S...... 2 1 H. Analytical Procedures ...... S...... 23 I. SEM Studies ...... 23
III. RESULTS ...... S...... 25 A. Algae Populations ...... S...... 25 B. Laboratory Culture ...... 26 C. Metal Levels in Field-Collected Algae...... 30 D. Uranium Toxicity ...... 0 34 " 0 E. 24-Hour Uptake ...... 37 F. Long-Term Uptake ...... i 41
IV. DISCUSSION ...... 54
APPENDIX A. Analytical Procedures ...... 63
APPENDIX B. Statistical Test Values ...... 66
REFERENCES ...... 68 Sii
ACKNOWLEDGEMENTS
My foremost thanks go to my adviser, Dr. James Brierley, for his patient, continued encouragement of this work and for his many suggestions for lines of research and methods for carrying them out. I thank my committee members,
Corale Brierley and Dr. Donald Brandvold, for their help ful discussions and for their interest in the research.
Dr. Thomas Lynch's help with the pond productivity calcu lations and with the statistical methods is greatly appreciated. My special thanks go to Barbara Popp,
John Hagstrom and J. D. Murray, at the New Mexico Bureau of Mines and Mineral Resources, for meticulously performing so many of the quantitative analyses. James Cleveland,
Chief Environmentalist for the Kerr McGee Corporation, provided the opportunity to sample the treatment pond
system.
This work was supported by the New Mexico Water Resources
Research Center grant 1345639 and by the United States
Bureau of Mines contract #J0295033, subcontracted from the
Center for Environmental Sciences, University of Colorado
at Denver, directed by Dr. Willard R. Chappell. ii i
ABSTRACT
Uranium mine wastewaters at the Kerr McGee Corporation site near Grants, New Mexico, are treated in part by retention in a series of ponds which support a mixed bacterial and algal flora. The pond algae, predominately
Chara, Spirogyra, and Oscillatoria, accumulate uranium and molybdenum from minewater. In Chara, average levels were
440-580 ppm uranium and 0-22 ppm molybdenum. Levels in
filamentous algae varied over the year from 1090-24?0 ppm uranium and 29-260 ppm molybdenum; the lower values were observed in summer and the higher in winter. In labora tory studies, Spirogyra removed more uranium and molybde num from solution than Chara in 24-hour uptake. Grinding
Spirogyra cells increased removal of uranium and molybde
num 2- to 3-fold over whole cells, with up to 82" removal
of uranium and 37' removal of molybdenum. In long-term
studies involving decaying algae in contact with sediment,
the addition of algae to sediment promoted reducing
conditions and usually increased sediment retention of
uranium and molybdenum, but removal was reversible unless
the model system contained high levels of organic
material and sediment.
In the present algae pond system, the mass of algae
is insufficient. 4o remove significant levels of uranium
and molybdenum from the high volume of minewater. .1\V
Increasing pond productivity would not produce enough algae to reduce the present levels of uraniurm and molybdenum by algal adsorption alone. However, greater productivity might enhance removal by increasing the amount of organic substrate available for sulfate-reducing bacteria, thus promoting hydrogen sulfide production in pond sediments, which would enhance trace contaminant removal. LIST OF TABLES
Table Page
I. Chemical Parameters in Pond Water and Sediment ...... 12
II. Media Used in Laboratory Culture of Algae ...... 15
III. Conditions of 24-Hour Experiments ...... 20
IV. Conditions of Long-Term Experiments ...... 22
V. Test for Recovery of Uranium and Molybdenum in Digestion of Algae ...... 31
VI. Uranium and Molybdenum Levels in Field-Collected Algae ...... 32
VII. Uranium and Molybdenum Levels in Field Collected Algae Averaged over Ponds 1, 2 and 3 ...... 33
VIII. Growth of Algae in Increasing Concentrations of Uranium ...... 35
IX. 24-Hour Uptake of Molybdenum and Uranium by Chara and Spirogyra...... 39
X. Long-Term Experiment I: Uptake of Uranium and Molybdenum by Spirogyra ...... 43
XI. Long-Term Experiment II: Uptake of Uranium and Molybdenum by Chara in Presence of Sediment ...... 45
XII. Long-Term Experiment III: Uptake of Uranium and Molybdenum by Chara and Spirogyra in Presence of Sediment ...... 48
XIII. Long-Term Experiment IV: Uptake of Uranium and Molybdenum by Chara and Sediment in Dark-Maintained Cultures ...... 51
XIV. Eh Measurements for Long-Term Experiment IV Chara and Sediment in Dark-Maintained Culture.. 53
XV. Long-Term Experiment IV: Percentage of Uranium and Molybdenum Removed by Chara and Sediment in Dark-Mainained Culture ...... 53 vi
LIST OF FIGURES
Figure Page
1. Schematic Representation of Pond System ...... 11
2. Chara. SEM micrograph ...... 27
3. Spirogyra. SEM micrograph ...... 28
4. Oscillatoria. SEM micrograph ...... 29 I. INTRODUCTION
A. Bioaccumulation of Heavy Metals
Wastewaters from industrial and mining operations often contain heavy metal pollutants in quantities too small to warrant recovery by conventional extractive techniques, but which must be removed to maintain water quality stand ards. Uranium minewaters, for example, may contain dilute concentrations of uranium, molybdenum, selenium, radium, and other associated ions (Brierley and Brierley, 1980).
Lead mines discharge waters with low levels of lead, zinc, copper, cadmium and manganese (Gale and Wixson, 1979), and the zinc industry discharges zinc, cadmium, copper, and mercury in its effluents (Jackson, 1978). The possibility of using algae for water treatment has been considered as a low-cost alternative for removing such mixtures of metals, as the ability of algae to accumulate a range of heavy metals has been recognized (Jennett, Iassett, and Smith,
1979).
The bioaccumulation of heavy metals by microorganisms has been investigated extensively in relation to environ mental pollution (Gadd and Griffiths, 1978; Leland et al.,
1978). Analyses of algae collected in contaminated waters show concentrations of metals 102 to 104 times higher than in surrounding waters (McDuffy et al., 1976; Ray and White,
1976; Trollope and Evans, 1976). Good correlations between concentrations of copper and lead in seawater and benthic algae were found by Seeliger and Edwards (1977), who suggested that algae might be used as indicators of environmental pollution. However, changes in the density and structure of naturally fluctuating populations, as well as physical and chemical differences in algal species, often obscure the relationship between concentrations in solution and concentrations in algae (Briand, Trucco, and Ramamoorthy, 1978; Knauer and Martin, 1973).
Canterford, Buchanan and Ducker (1978) reported that concentrations of zinc, cadmium and lead in a marine diatom increased with higher external concentrations, but that the percentage of metals removed from water decreased at the higher concentrations. Coleman, Coleman and Rice
(1971), using concentrations of up to 35 ppm zinc and
3 ppm cobalt, reported increased percentage uptake by freshwater algae with increasing concentrations of metals.
The study of microbial bioaccumulation in respect to treatment of polluted waters has been given increasing attention (Kelly, Norris and Brierley, 1979). Several water treatment systems have been developed to take advantage of the metal concentrating ability of algae.
Gale and Wixson (1979) have investigated an artificial stream meander system which has beon successful in remov ing lead, zinc and copper from lead mine effluents.
Evidence indicated that the mixed algal flora, including
Cladophora, Rhizoclonium, IHydrodictyon, and Spirogyra, entrapped suspended mineral particles and accumulated metals by a cation exchange process. Investigation of zinc mine and smelter wastewaters by Jackson (197S) showed that planktonic algae blooms, stimulated by sewage effluents, removed zinc and some cadmium and copper from solution. A system of sequential ponds was suggested to maximize contact with the algae and to encourage sedimentation of dead algae so that accumulated
metals could be sequestered by bacterially produced sulfides in the sediments. Filip et al. (1979) utilized a combined sand filtration and algae bioaccumulation system to remove copper, cadmium and chromium in waste water primary treatment lagoons. Populations of Chlorella,
Scenedesmus and Oscillatoria removed 70 to 90' of the
1.5 ppm cadmium and 2.3 ppm copper in solution, with 9S to
100% removal achieved in combination with the sand filter.
A lower percentage uptake of 20. was observed for 15 ppm chromium in solution, and no increased removal was effected by the sand filter. Filip et al. (1979) postulated that binding sites for chromium had been saturated at the higher concentration.
B. Mechanisms of Uptake of Heavy Metals
Two types of mechanisms for uptake of heavy metals have been recognized in microorganisms: intracellular transport and binding of metals to cell surfaces. Intra cellular uptake of heavy metals by energy-dependent I
transport is metabolically controlled and utilizes pathways which exist for cellular reguration of trace metals and other essential ions, such as the K Ca and Mg++ transport systems, or the sulfate permease system which can transport oxyanions. The binding of metals to cell surfaces, on the other hand, involves the physical-chemical processes of adsorption and ab sorption. Kelly, Norris and Brierley (1979) concluded that retention of sorbed ions depends on formation of stable metal-organic complexes on cell surfaces, deposition of insoluble forms of the metals in cells, and incorporation of metals into cellular constituents.
Metabolically mediated uptake of several heavy metals has been demonstrated in algae. 7inc uptake in
Chlorella fusca was characterized by initial rapid, reversible adsorption followed by long-term energy dependent, largely irreversible uptake into the cell
(Matzku and Broda, 1979). Similar patterns held for thallium and cadmium transport in Chlorella fusca
(Solt, Paschinger and Broda, 1971; Mang and Trombella,
1978). Uptake of thallium may follow the pathway for potassium uptake, while cadmium seems to be taken up by the magnesium transport system. Manganese has also been shown to compete with cadmium uptake (Hart, Bertram and Scaife, 1979).
Nonmetabolic uptake of heavy metals plays an important 5
role in their bioaccumulation by algae. Algae have an overall negative charge (Bayne and Lawrence, 1972) which aids in binding cations to the cell surface. Veroy et al. (1980) found that lead and cadmium binding in the marine alga Eucheuma could be accounted for entirely by the formation of metal complexes with the negatively charged cell wall polysaccharide carrageenan. In a study of localization of lead in field-exposed and laboratory test Cladophora, Gale and Wixson (1979) found that differences in lead accumulation on cell walls depended on length of exposure. Lead deposits were detectable on the surface of filaments when the cells were exposed
to high concentrations of lead over a period of hours in
laboratory tests. However, field-exposed specimens and
laboratory specimens grown in lead solution over a period
of weeks showed deposits only within the cell wall and
capsular material, and none on the cell surface. The
contrast between short- and long-term exposure suggests
that an exchange absorption was involved in the incorpor
ation of lead into cell wall constituents, while adsorp
tion to the surface'accounted for short-term uptake.
Extrace]lular material can also accumulate heavy
metals. In the semicolonial marine alga Phaeocystis,
whose individual cells are bound by a slime matrix, zinc
accumulated in the colony matrix but was substantially
released on disruption of the colonies, while manganese 13
was taken up and retained extracellularly (Morris, 1971).
Hodge, Koide and Goldberg (1979) have suggested that the organic extracellular coating of marine macroalgae was involved in retention of particulate uranium, polonium and plutonium.
The phenomenon of increased adsorption of metals in dead over living cells has been observed in marine macro algae for zinc but not cesium (Gutknecht, 1965), in marine phytoplankton for thallium but not potassium
(Cushing and Watson, 1968), and in Chlorella regularis fpr UO 2 (Horikoshi, Nakajima and Sakaguchi, 1979).
Horikoshi et al. (1979) suggested that the increased uptake of UO2 in the heat-killed Chlorella regularis was due to an increase in adsorption sites on the disrupted cell walls rather than to an inhibition of cellular regulatory processes, since whole cells treated ++ with metabolic inhibitors adsorbed UOJ2 at the same
lower levels as the metabolizing whole cells. Adsorbed uranium was associated with cell wall and cell membrane
fractions. Ferguson and Bubela (1974) used suspensions
of fresh particulate material from Chlorella, Ulothrix
and Chlamydomonas to accumulate copper, lead and zinc.
At higher concentrations of metals, formation of metal
organic complexes produced deviations from monolayer
adsorption behavior, so that higher percentages of metals were removed from solution. The ability of the particulate 7
suspensions to accumulate metals was considered good evidence for the ability of algae to in'fluence the
formation of metal-rich sediments.
C. Algae, Metal and Sediment Interactions
In pond and stream systems, the retention of heavy metals accumulated by either living or dead algal material
depends on formation of insoluble metal-organic complexes
and, perhaps more important, on the interactions between
metals, algae and sediment.
Algae cells come in contact with sediments primarily
as decomposing material. In the water treatment system
studied by Jackson (1978), dying algae sank to the bottom,
carrying accumulated metals into the sediment. Hydrogen
sulfide produced by bacteria during decomposition of the
algae could sequester the metals as insoluble metal
sulfides. Zinc, copper, cadmium and mercury were associated
more strongly in the sediments with sulfide than with
organic carbon.
Studies on aerobic and anaerobic decomposition of a
wide range of algae types indicate that both processes
proceed at about the same rate and result in mineralization
of around 60% of cell material, while the remaining fraction
persists as particulate material with similar carbon content
to the original cells (Fallon and Brock, 1979; Foree and
McCarty, 1970; Seeliger and Edwards, 1979; Ulen, 1978).
This refractory material could be a source of organic
material for long-term complexing of metals in sediments. 8
Relatively few studies on algal bioaccumulation of heavy metals have considered the fate of the metals as the algae die and decay. Filip et al. (1979) observed release of 66% of accumulated cadmium and copper and 53% of chromium in decaying algae cultures after one week of exposure to the metals in growing cultures. Jennett, Smith and Hassett
(1979) noted slow release of lead from dead or dying
Chlamydomonas cells after a short accumulation period; mercury accumulated by Nostoc showed no release. Seeliger and Edwards (1979) followed the release of copper in decomposing red marine algae. During the decomposition period of 100 days, 30 to 60% of the copper accumulated previously be the growing algae was retained in refractory material and in particulate detritus derived from the decay ing algae. The remaining fraction of accumulated copper was released primarily in dissolved organic material and to a lesser extent as inorganic material. While the inorganic copper was available for readsorptionby algae, the organic copper complexes were not taken up by algae. Such dissolved metal-organic complexes may render the metal less suscept ible to bioaccumulation (Sunda and Lewis, 1978) and, in some cases, may increase the solubility of the metal from a bound form (Bloomfield and Kelso, 1973; Jenne and Luoma,
1977).
In a study on metal, algae and sediment interactions,
Laube, Ramamoorthy and Kushner (1979) observed that after uptake of cadmium and copper by Anabaena, cell lysis 9
released the metals as organic complexes of high molecular weight. Whether these organic complexes could be readsorbed
onto the sediment was not investigated. It was noted,
however, that Anabaena cells accumulated metals far more
readily from water than from sediment, based both on final
levels of the metals derived from each source and on per
centage of available metal removed from each compartment.
D. Objectives of Present Study
The Kerr McGee Corporation in the Ambrosia Lake District near Grants, New Mexico, has developed a treatment system for uranium mine wastewaters. Water from two underground mines is pumped through a series of settling and algae ponds, which support a mixed bacterial and algal flora.
Concentrations of uranium, selenium, and molybdenum in the water and sediments of the pond system have been established
(Brierley and Brierley, 1980).
The studies described in this thesis have been under taken to identify the major algal populations in the ponds and to determine the extent of uranium and molybdenum accumulation in the algae in situ . Also, laboratory experiments have been conducted to study the ability of two selected algae, Chara and Spirogyra spp., to accumulate uranium and molybdenum on short- and long-term exposure, with emphasis on the long-term retention of the metals in the presence of pond sediment. 10
II. MATERIALS AND METHODS
A. Site Description
The wastewater treatment system consists of a series of ponds which receive water pumped from two underground mine sections at a rate of 2x106 gallons a day per section
(Figure 1). Barium chloride is added to water from both sections prior to entry into the algae ponds, in order to coprecipitate radium as radium sulfate. Section 35 water passes through an ion exchange plant to remove uranium prior to combining with section 36 water. The residence time for the combined waters in the three algae ponds is about 2.5 days, based on a volume of 9x106 gallons
(1.25xi05 ft 3) in the three algae ponds divided by the daily input of 4x106 gallons.
Table I gives average values for water and sediment parameters measured between 11/15/78 and 4/29/80 (Brierley and Brierley, unpublished data).
The pond sediments provide a reducing environment (Eh,
-350 mV) for a large population of sulfate-reducing bacteria of the genera Desulfovibrio and Desulfotomaculum
(Brierley and Brierley, 1980). In the summer months between June and September, the pond perimeters support populations of Typha, Juncus, and sedges, which die back
in the winter months. 11
Section 35 Mine Secti'on 36 Mine A "*A100' 200' I IA- B-I
-O I' ) I B-oI IN ~0 '4-5", oý30
0 q-Q
Discharge 160'
Figure 1. Schematic Representation of Pond System 12
TABLE I CHEMICAL PARATERS IN POND WATER AI•D SEDIENT
LOCATION pH 0ONDUCTIVITY SUSPENDED SOLIDS NO 3 (pmhos) (ppm) (ppm) x s.d. x s.d. x s.d. x s.d. S-35 minewater 7.37 0.71 1280 225 129 113 1.10 0.50 A-i sediment A-2 sediment A-2 effluent 7.56 0.63 39 23 1.10 0.46 A-3 sediment A-3 effluent 7.58 0.56 21 12 0.97 0.35 A-4 sediment A-4 effluent 7.60 0.48 1290 240 22 9 1.08 0.45
S-36 minewater 7.60 0.02 1320 220 266 243 0.60 0.64 B-i sediment B-i effluent 7.19 0.30 1350 200 21 27 1.07 1.33 B-2 sediment B-2 effluent 7.76 0.51 1290 150 20 21 0.46 0.56 algae influent 7.67 0.47 1290 180 67 129 0.44 0.16 alg-1 sediment alg-1 effluent 7.66 0.30 1280 170 20 16 0.50 0.20 alg-2 sediment alg-2 effluent 7.68 0.46 1330 210 16 24 0.44 0.15 alg-3 sediment alg-3 effluent 7.81 0.45 1290 240 17 20 0.47 0.26 n=6 n=5 n=7 n=6
Pond B-I dry after 6/29/79 13
TABLE I--continued CHEIICAL PARA[ETERS IN POND WATER AND SEDIENT
LOCATION U(ppm) Mo(ppm) Se (ppm) SO4 (ppm) x s.d. x s.d. x s.d. x s.d. S-35 minewater 4.6 1.2 2.0 0.5 0.026 0.014 720 50 A-1 sediment 918 200 202 226 0.105 0.084 3900 560 A-2 sediment 882 262 105 101 0.091 0.061 3000 190 A-2 effluent 5.1 0.:5 1.9 0.5 0.017 0.005 720 50 A-3 sediment 1390 474 520 609 0.090 0.050 9100 2500 A-3 effluent 5.0 0.5 1.9 0.5 0.045 0.064 740 100 A-4 sediment 1634 857 420 679 0.063 0.037 4600 2600 50 A-4 effluent 4.4 0.8 1.9 0.4 0.022 0.007 690
680 60 S-36 minewater 3.4 3.4 0.04 0.05 0.014 0.023 2500 B-I sediment 3128 2595 25 64 0.041 0.053 3500 B-I effluent 1.1 0.6 0.04 0.02 0.010 0.013 720 30 B-2 sediment 1488 1493 70 84 0.039 0.024 4100 400 B-2 effluent 1.0 0.7 0.05 0.02 0.005 0.004 680 60
700 50 algae in fluent 0.9 0.5 0.9 0.3 0.009 0.003 2200 alg-1 sediment 1512 643 272 160 0.064 0.038 10800 700 50 alg-1 effluent 1.0 0.7 1.0 0.3 0.011 0.003 12400 alg-2 sediment 963 386 298 493 0.026 0.015 23700 700 50 alg-2 effluent 0.8 0.4 0.9 0.3 0.010 0.004 12300 2500 alg-3 sediment 1097 283 371 131 0.073 0.042 700 50 alg-3 effluent 0.7 0.3 0.8 0.4 0.010 0.003 n-6 eff n-8 n=7 eff n=7 sed n=6 sed n=4
*Pond B-1 dry after 6/29/79 water and total S (ppm) in sediment **Values represent S04(ppm) in 14
B. Collection Procedure
Algae samples were collected from rll three algae ponds on 9/25/79, 1/19/80, and 4/29/80. Samples were collected from the edge of the ponds, given a preliminary wash in pond water to remove excess particulate material, and stored in nonsterile polyethylene bottles on ice for trans port to the laboratory. After further washing in deionized water, the samples were either dried for metal analyses or transferred to aquaria for laboratory culture. The most prominent algae were identified to genus using the keys of
Prescott (1954) and Smith (1950).
C. Laboratory Culture
The algae were grown in mixed culture in 20 1 aquaria.
Shen's medium (Shen, 1971) was generally used for culture, -1 with 0.2 g 1 NaHCO 3 added to Chara cultures (Forsberg,
1965). About 1% activated charcoal was added to the media to remove possibly toxic growth byproducts (Shen, 1971).
The algae were grown without aeration.
Table II gives nutrient concentrations for Shen's medium
as well as for other media used in laboratory tests.
Illumination was provided continuously (Forsberg, 1965) by 2 22' GE "Gro and Sho" f]oiirescent lights placed 12"
from the water surface, giving "5-40 fc illumination. The
temperature, which ranged from 180 C to 050C, was usually 230C.
D. Algae Digestions
In order to determine metal content in field-collected 15
TABLE I I
NEDIA USED IN LAR)RATORY CLnLTUR- OF ALGAE
1. Modified Bristol's Medium concentration an-unt stock in medium stock solution solution used 1-1 NaNO3 0.25 g 10 g/ 400 ml 10 ml 1-1
CaCl2 *2H 2 0 0.25 10 g/ 400 ml 10
MgSO4"7H2 0 0.075 3 g/ 400 ml 10
K2 HPO4 0.075 3 g/ 400 ml 10 10 KH2 PO4 0.018 0.7 g/ 400 ml NaCI 0.025 1 g/ 400 ml 10 1-1 H FeSO4-7H20 in 1 ml 2so4 2.5 g/1000 ml 1
2. Bold Basal Medium
10 ml 1-1 a. NaNO 3 0.25 g 1-1 10 400 ml 10 CaCl 2 " 2H20 0.025 1 400 ml
MgSO4 7H2 0 0.075 3 400 ml 10 K2HFX)4 0.075 3 400 ml 10 K11HP0 2 4 0.18 7 400 ml 10 NaCl 0.025 1 400 ml 10
b. EDTA 0.05 50 g/1000 ml KOH 31 g 1 c. FeSO4 "7II2 0 0.005 4.98g/1000 ml 1 ml 1
d.H 3 BO4 0.011 11.42g/1000 ml
e. micronutrient solution ZnS4 *71120 8.82g/1000 ml
MnC1 2 " 4H20 1.44 1.57 CuSO4 51H20
Co(NO3 ) 2 "6H20 0.49 16
TABLE I I--continued
MEDIA USED IN LABORATORY CULTJRE OF ALGAE
1. Shen's Medium concentrat ion in medium
g 1-1 co(NH2 )2 0.04 0.10 g CaC1 2 21120
''SO 4 . 7Ho20 0.10 g
Na2CO3 0.02 g 0.01 g NaSiO3 K2HPO4 0.6 mg KCI 0.05 g Tris 0.50 g ml each 1-1 stock solutions b, c, d, and e (Bold Basal), 1
*9 Stein (1.971) Shen (1971) 17
algae, samples were prepared for uranium and molybdenum analysis by digestion with nitric acid'and hydrogen peroxide.
Samples of 0.1 to 0.75 g dry weight were added to 10 ml
0 concentrated HNO . After overnight incubation at 85 C, the digestion was completed by addition of 5 ml 30% H202 with heating at 135 0 C for 5 minutes. Samples were cooled, filtered through 0.45 pm Millipore filter and brought to
250 ml volume with distilled water. Blanks without algae were run as controls for the digestion procedure.
A test for recovery of the metals by the digestion process was run on samples of 0.25, 0.5, and 0.75 g air dried Chara (9/25/79 collection), half of which were spiked with uranium (UO 2 So 4 3H 20) and molybdenum (Na 2 Moo 4 "2H 20) to produce 0.2 ppm concentrations of each metal in the final dilution volume. The unspiked samples were compared to the spiked samples to evaluate recovery of metals added to the algae.
E. Uranium Toxicity Test
While uptake studies in the laboratory initially were
conducted using uranium and molybdenum concentrations close
to those in the minewaters, higher concentrations were
desirable to facilitate quantitative analyses of the metals.
A toxicity test for uranium was run to determine a range
of concentrations which could be used without interferir; with algal growth. 18
Two series of concentrations were prepared, one using pond water collected from the algae ponds and filtered through 0.45 pm Millipore filter, and one using Shen's medium without EDTA or Tris. Tris and EDTA were omitted because of possible chelation effects which might mask toxicity (Good. et al., 1966; Sunda and Lewis, 1978).
Concentrations of uranium used were 0, 10, 20, 50, and
100 ppm. 5 ppm molybdenum were added to the media in all but the flasks containing 0 ppm U. Molybdenum and uranium spikes were added to batches of media and the pH adjusted to 7.5 with 0.1N NaOH before dispensing so that each series of flasks at a given concentration contained the same preparation.
100 mg wet weight of Chara, Spirogyra or Oscillatoria were added to 50 ml media in 250 ml Erlenmeyer flasks, which were incubated in a 12 hour light/dark regime for two weeks.
Growth of algae was monitered by visual comparison of flask contents. The p1l was checked after I week.
Flasks without algae but containing the series of uranium spikes were kept under similar conditions and analysed for uranium and molybdenum content at the end of the test.
F. 2 4 - Hour Uptake of Molybdenum and Uranium
Removal of molybdenum and uranium from solution by algae was tested in 24-hour uptake experiments. Table III 19
summarizes the specific conditions under which each experi
ment was conducted. In each test, the'algae were washed
in deionized water, dried on paper towelling, and weighed
out immediately. The wet weight in Table III is the amount
actually weighed out, while the dry weight was taken from
a representative aliquot of the algae being tested, which was dried overnight at 85 0 C and reweighed. Particulate
material was produced by grinding the algae one minute in
a Sorvall Omni-Mixer, in a modification of a procedure
suggested by Ferguson and Bubela (1974). Suspensions were
used immediately instead of being frozen, and suspensions
generally were not centrifuged before use. In experiment
1, one set of Chara particulate suspensions was centrifuged
at 14,500 rcf and the pellet material alone used for uptake,
and one set was left uncentrifuged.
To prepare test flasks, media were dispensed into the
Erlenmeyer flasks and the pH adjusted with iN NaOH or iN HCI.
After the algae were added, the flasks were spiked with
stock uranium (UO 2 SO4 "311 2 0) and molybdenum (Na2MoO 4 -211 2 0)
solutions to produce the required concentrations, and the
pH readjusted. Flasks without algae were used as controls.
The experiments were carried out in stationary flasks
incubated 24 hours in light.
The amounts of metals remaining in solution were deter
mined after filtering the media through 0.45 im Millipore
filters to remove algal material. It was necessary to 20
TABLE III
ODNDITIONS OF 24-HOUR EXPERIMErs
EXPT. PPM U PPMf O ALGAE MIT. ALGAE MEDIA AMT. MEDIA PH 1:10 Bristol's 200 ml 7.7 1 5 3 Chara 2.0 g
(0.4 g)
1:10 Bristol's 200 ml 7.7 2 10 3 Spirogyra 2.0 g
(0.2 g)
or 7.8 3 10 2.5 Chara 1.0 g Shen's 100 ml 100 (0.2 g) pond water ml 7.8
Shen's or 100 ml 4 10 5 Spirogyra 1.0 g 8.0 8.0 (0.1 g) pond water 100 ml
wet weight
dry weight 21
centrifuge the particulate suspensions for 10 minutes at
4,340 rcf before filtering. In experiMent 3, the algal material was recovered and analysed for metal content.
G. Long-Term Studies
Long-term interactions between water, algae and sediment were modeled in the laboratory using 2 1 culture vessels containing 1 1 medium spiked with uranium and molybdenum, and growing algae cultures. Table IV summarizes conditions in four long-term experiments. Algae were added in amounts measured by wet weight, with comparable aliquots dried for the dry weight determination. Growth was monitered visually. The first three experiments involved a period of growth in light for the algae exposed to the metal solutions.
In experiments II and III, the light incubation period was followed by a long period in the dark, during which two of the algae containers and two of the control containers were given additions of pond sediment. In experiment IV, the amounts of sediment and algae were increased relative to the amount of solution, and the light incubation period was omitted since the algae were added as dry material.
The pH of all tests was monitered, and Eh measurements were taken during the latter period of experiment IV.
Concentrations of uranium and molybdenum in the media were monitored by periodic removal of 100 ml solution, which were filtered and acidified before metal analysis. The
100 ml aliquot was replaced with an equal volume of medium TABLE IV
CONDITIONS OF LONG-TERIM EIPERIMENTS
DAYS DAYS MrT. IN DARK SEDIMENT EXPT. PP,I U PPM O0 ALGAE AM.r. ALGAE MEDIA PH IN LIGHT *• I 5 2 Spirogyra 15 Bristol 's 6.5 14 0 0 II 10 3 Chara 10 g 1:4 Bristol's 7.7 12 142 150 g (2.4 g ) 152 150 g III 5 2 Chara 5g Shen's 7.8 14
(1.1 g) L' L,I Spirogyra 5g (0.4 g) IV 20 5 Chara 20 pond water 8.1 0 190 1000 g
wet wei-ght dry weight added as dry material 23
containing the initial metal concentrations.
H. Analytical Procedures
Uranium concentrations indigested algae or in water samples were determined colorimetrically by the TOPO extraction procedure (Johnson and Florence, 1971). Repli cate analyses for a given sample varied from 0 to 33% with a mean variation of 9% (based on 12 samples).
Molybdenum was assayed colorimetrically by the thiocyan ate procedure (Meglen and Glaze, 1973). The analysis varied from 1 to 35% with a mean variation of 20% (based on 5 samples). Appendix A gives a detailed description of both the uranium and the molybdenum analyses.
Water samples from uptake or toxicity studies were generally diluted 1:10 or 1:20 before analysis, while digested algae samples were run at 1:2 or at no dilution.
I. SEM Studies
Algae specimens were prepared for microscopy by dehyd ration in the following ethanol:amyl acetate series: ethanol in distilled water 30%, 50%, 70%, 95%; 100% ethanol twice; amyl acetate in absolute ethanol 30%, 50%, 70%, 95%;
100% amyl acetate twice. Samples were left in each solution
15 minutes (Hayes, 1973). The samples were transferred to liquid carbon dioxide in a Ladd Critical Point Dryer for critical point drying. The dried samples were mounted onto stubs using nitrocellulose glue and were sputter-coated with carbon in a Denton Vacuum (DV-502) high vacuum 24
evaporator. The specimens were examined with a Hitachi
HHS-2R scanning electron microscope. .
Micrographs were taken on Polaroid Type 55 positive/
negative 4x5 Land film. 25
III. RESULTS
A. Algae Populations
The predominant algae in the ponds were the macrophytic green alga Chara, the filamentous green alga Spirogyra, and the filamentous blue-green alga Oscillatoria. Rhizoclonium, another filamentous green alga, was found at the inflow of algae pond 1 during the summer. Unidentified diatoms and unicellular green algae were associated with the floating mats of filamentous algae and with Chara; these associated algae were most abundant in the summer months.
The composition of the algal flora varied over the year. In the winter months of November, January and March, growth of algae was sparse. The floating algae were con centrated at the inflow and outflow of each pond.
Oscillatoria tended to dominate the filamentous algae in the winter. Collections from April, June and September showed a higher proportion of Spirogyra. During the summer months, growth of the filamentous algae extended in patches out into the deeper sections of the ponds, although only pond 3 showed extensive coverage even in summer.
Chara formed a dense mat of fronds over the pond floors during the entire year, but growth evidently took place mainly in the summer months. Winter vegetation of Chara was weathered and more mineralized than summer vegetation, suggesting that the winter material was a survival of summer growth. 26
Figures 2, 3 and 4 are scanning electron micrographs of
Chara, Spirogyra , and Oscillatoria, r~spectively. The
epiphytic diatoms are especially evident on Chara. Also,
the heavy mineral coating on Chara can be seen, in contrast
to the smooth filaments of Oscillatoria and Spirogyra.
B. Laboratory Culture
Algae cultures were grown initially in modified
Bristol's medium (Table II), chosen because of its general applicability to a wide range of green algae. Repeated
failure of Chara and limited growth of Spirogyra led to trials with other media.
Additions of trace elements and EDTA as specified in
Bold Basal medium (Table II) did not improve Chara or
Spirogyra growth and seemed detrimental to Oscillatoria.
A unialgal culture of Oscillatoria was maintained for a year in modified Bristol's medium but would die when trans ferred to Bold Basal medium.
The medium used successfully for Chara and Spirogyra culture was Shen's medium (Table II), developed specific ally for Chara based on the alga's requirement for extremely low phosphorus concentration (Shen, 1971).
Forsberg (1965) has reported that phosphorus concentrations above 25 pg 11 are inhibitory to Chara. Collections of
Chara and Spirogyra transferred from field collections directly to aquaria containing Shen's medium could be maintained three to four weeks providing the initial - Adiýa OW
4w 28
Figure 3. Spirogyra, scanning electron micrograph (X875) 29
Figure 4. Oscillatoria, scanning election micrograph (X1575) 30
aliquot was sparse. Subsequent overgrowth of other algae in the aquaria replaced Spirogyra and Chara. A typical succession of growth in an aquarium initially stocked with
Chara consisted of Oscillatoria on the aquarium walls and a surface network of blue-green Lyngbya filaments mixed with unicellular and colonial green algae. Festoons of filamentous Ulothrix appeared after several weeks.
Although Chara had grown well in the aquarium initially, after growth of the successional algae only a few ptiolated shoots were formed. The cultures of Chara and Spirogyra used for laboratory tests were removed from the aquaria before this stage of overgrowth had begun.
C. Metal Levels in Field-Collected Algae
Table V gives results for the test for recovery of metals in the digestion procrss which was described in section II D. Molybdenum was less likely to be recovered in the analysis than uranium. The inability to recover the spike amount of molybdenum was evident in solutions both with and without algal material and probably was due to loss by precipitation or adsorption onto flask walls rather than to incomplete digestion of the algae. Recovery of uranium was fairly consistent over the range of sample sizes and was unaffected by the addition of the uranium spike.
The -esu]ts of analyses for three collections of algae
are summarized in Table VI. Table VII gives the means for 31
TABLE V
TEST FOR RECOVERY OF URANIUM AND .MOI,3)ME•71.M IN DIGESTION OF ALGAE
ALGAE SAMPLE PPM U, PPM U PPM TU ** PPM .1W PPM MD PPM•. :M ** DRY WEIGHTr SPIKE IN SAMPLE IN ALCAE SPIKE* IN SA!MPLE IN ALGAE 0g 0.2 0.29 0.2 0.13 25 g 0.2 0.62±0.06 420 0.2 0. 1±o.0 0 25 g 0 0.34-0.10 3-40 0 0 0 50 g 0.2 0.99 400 0.2 0.!20 0 50 g 0 0.55±0.22 275 0 0.01-0.00 5 75 g 0.2 0.95±0.06 250 0.2 0.10±0.09 0 75 g 0 0.81±0.05 270 0 0.06±0.04 20
Samples were spiked before digestion with uranium =-mi mloybdenu-m or were left unspiked. Concentrations in algae were calculated by the forimla (ppm sample - ppm spike) x 0.25 1 x t0 ppm in algae g algae 32
TABLE VI UPANIUM AND M)LYt)ENUMI IEES IN FIED-CX)LB= ALGAE
DATE OF cCO-rION: 9/25/79 1/24/80 4/29/80 A. URANIUM (ppm) Chara pond 2 544-439 (7) 580 (1) 375±-25 (2) pond 3 330±285 (2) NC 580 (1) Spirogyra, Oscillatoria pond 1 NC 1850±400 (4) 2015±15 (2) pond 2 870±90 (3) 2670±530 (4) 1700±420 (?,) (1) pond 3 NC 3100±300 (2) 1150 Rhizoclonium pond 1 1420±50 (2) NC 1700±100 (2)
B. .OLYITDEN.I (ppm) Chara pond 2 22 ±20 (2) 0 (2) 40±4 (2) pond 3 0 (2) NC 4±0 (2) Spiropyra, Oscillatoria pond 1 NC 320-150 (4) 70±17 (2) pond 2 27±16 (3) 270±200 (4) 53±8 (4) pond 3 NC 120±50 (2) ,35±1 (2) Phizoclon itum pond 1 31-+1 (2) NC 30±11 (2)
Number in parenthesis indicates sample size NC = no collection 33
TABLE VII. URANIUM AND MDLYEDJE.IM LEVELS IN FIELD-ODLRTE ALGAE AVERAGED OVER PONDS 1, 2, AND 3
DATE OF COLLECTION: 9/25/79 1/24/80 4/29/80 U PPM M PPM U PPM M PPM U PPM M PPM Chara 500±390 16±19 580 0 440±120 22±21 Spirogyra Oscillatoria 1090±310 29±12 2430±670 260±190 1710±350 48±18 Rhizoclonium
filamentous algae and for Chara calculated over all samples
from each collection date, taking the three ponds as one
collection. Levels of molybdenum and uranium were higher
in filamentous algae than in Chara by 2 to 3 times for uranium and about 2 times for molybdenum. Wide variation
in both molybdenum and uranium levels were found; uranium
ranged from a low of 330 ppm (Chara, 9/25/79) to 3100 ppm
(filamentous, 1/24/80), and molybdenum from undetectable
levels in some Chara samples to a high of 320 ppm in the
filamentous algae collected 1/24/80. Generally, in a given sample the uranium concentration was an order of magnitude higher than the molybdenum.
Some seasonal variation was seen in the filamentous
algae but was not obvious in Chara. A one-way analysis of variance for uranium and molybdenum levels in filamentous
algae by month showed significant variation at the .01
level (see Appendix B for degrees of freedom and F values,
following the method of Snedecor and Cochran, 1967). The
levels of uranium were significantly different for all months, with levels increasing in the order September('79) 3-1
April ('80), January ('80). Molybdenum concentrations were significantly higher in January ('80) Cver September ('79)
and April ('80), but the difference between September and
April was not.significant. There was no correlation be
tween the metal content of a sample and pond from which it was collected.
D. Uranium Toxicity
Table VIII summarizes the growth response of Spirogyra,
Chara, and Oscillatoria to increasing concentrations of
uranium. After 1 day of culture, Oscillatoria trichomes
had spread out over the flask surface in pond 0 ppm and
10 ppm flasks, but not in Shen medium or in pond 50 ppm.
No differences were seen among the Chara or Spirogyra
flasks; even at 100 ppm, the cells were not lysed and
looked viable. By day 3, some Spirogyra cultures were
moribund (those marked with a minus sign in Table VIII),
and Chara internodal cells had lysed in Shen medium.
Oscillatoria cultures in pond water showed active growth
even at 50 ppm.
By day 11, all cultures in Shen medium were dead or
moribund. The medium had been modified by omission of
EDTA and Tris. which evidently rendered the medium unfit
for the algae, since even the cultures with no added
uranium died. There were no significant differences in
pH among the Sben medium flasks and the pond water, but
trace elements in the medium might have been toxic in the
absence of EDTA. 35
TABLE VIII GROIKMrI OF ALAE IN INCREASING CONCENTRATIONS OF URANIUM*
MEDIUM PPM U PM l.O ALCGE DAY 1 DAY 3 DAY 11 DAY 16 PH Shen's 0 0 Chara + + x x 8.0 Spirogyra + - x x 7.8
Oscillatoria - -- x x 7.9
Shen's 10 5 Chara + x x 8.0 Spirogyra + + x 7.5
Oscillatoria - - x x 7.5
Shen ' s 20 5 Chara + -- x x 7.9 + Spirogyra - x x 7.3
Shen's 50 5 Chara + + x x 8.0 Spirogyra + - x x 7.3
Oscillatoria - - x x 7.7
X Shen's 100 5 Chara + -- x 8.0 Spirog-ra + -- x x 7.3
+ + pond 0 0 Chara + + 8.2 water Spirogyra + + + + 8.6) Oscillatoria + + + + 8.2
+ pond 10 5 Clara + + + 8.5 water + + + Spirof'Yra + Oscillatoria + + + 8.3
+ pond 20 5 Cha!ra + + + 8.3 water Spirogyra + + + + 8.9 36
TABLE VII I--continued GROINWMM OF ALGAE IN INCREASING CONCEUTRATIONS OF URANIUM
ME•DIUMI PIPM U PPM•i Mo ALGAE DAY 1 DAY 3 DAY 11 DAY 16 PH + + + - 8.3 pond 50 5 Chara water Spirogyra + + + + 8.8 - + 4 8.5 Oscillatoria +
+ + x 8.2 pond 100 5 Chara water Spirogyra + + + + 8.7
Symbols represent good growth (+), poor growth (-), or dead cultures (x). 37
After 15 days' culture, Chara was still healthy in appearance in pond water at 0 ppm, 10 ppm, and 20 ppm uranium. Cultures at 50 ppm were moribund and those at
100 ppm were dead. Both Oscillatoria and Spirogyra maintained viable cells at the highest concentrations of uranium to which they were exposed. A microscopic examin ation of Spirogyra cells taken from the flasks revealed intact, well-formed cells containing fully-formed chloro plasts at 0 ppm, 10 ppm, and 20 ppm uranium. At 50 ppm and 100 ppm, chloroplasts in mid-strand, that is in cells which had grown before contact with the high uranium concentrations, were normal, but at the growing tips of the strands, chloroplasts showed evidence of disintegration.
Growth at the tips was characterized by abnormal cell division, giving rise to mid-strand side branches uncharac teristic of Spirogyra, and by abnormal cell wall formation.
Although cellular metabolism was not totally impaired at the higher concentrations, the abnormal cell growth sug gested that cultures could not be maintained at concentra tions much above 20 ppm uranium, and this is the concentra tion that was used in subsequent laboratory experiments.
E. 24- Hour Uptake
Uptake or uranium and molybdenum on a short-term basis was studied in Chara and Spirogyra in 24-hour laboratory uptake experiments. Results for the test- are given in
Table IX. Only one experiment (3A, Table IX) showed 38
detectable uptake of either uranium or molybdenum in
Chara. The detection of uranium and molybdenum in field collected specimens indicates that Chara does have the ability to accumulate the metals from solution, but the conditions of the short-term uptake studies evidently did not allow uptake or only at levels below detection.
The excess of uranium in the Chara preparations in
Experiment 1 (Table IX) probably is not due to release of uranium from the algae cells. Analysis of samples of
Chara before and after treatment gave 300 ppm uranium in the untreated algae, 930 ppm in the whole, 1950 ppm in centrifuged particulate, and 1190 ppm in particulate pre parations after incubation. Since the treated algae had higher levels of uranium than before treatment, it is more likely that the value for the control was low due to precipitation of uranium rather than that the algae had released uranium. Increasing the cell surface by disinti grationofChara cells had no effect on removal of metals from solution.
Spirogyra preparations adsorbed both uranium and molyb denum from solution (Table IX). In every case, disruption of the cells by omni-mixing increased the adsorption. The pattern of removal of both metals is strikingly similar in the three different solutions employed in the tests.
Disrupting the cells increased uptake of uranium ?- to 3 fold in each case. Increasing the external concentration TAPLE IX 24-IOUR UtfPAKE OF MO10 AND U BY CMARA AND SPIliJGYRA
LEPERIMET* PPM U % PDEOVED MG U/G ALGAE PPM MO %t REMOVED MG MO/G AIGAE 1 Chara in Bristol's Control 2.5±0.4 2.9-O. 0 Whole 18.4±2.0 0 3.0-0.0 0 Centrifuged Particulate 18.0-+1.3 0 2.8±o.1 0 Particulate 20. 3+0.4 0 '2.10.3= °33 0
.A Chara in Shen's C-, Control 10.1±0.1 2.2±0.1 M'ole 8.2±2.6 0.9 mg/g 2.7±0.4 0 Particulate 8.3±2.5 0.9 mg/ g 9.2±0. 2 0
3B Chara in pond water Control 9.7±0.4 2.4+0.1 Mhole 9.9+1.3 0 2.5+0.1 0 Particulate 10.0-1.1 0 2.5t0.2 0 TABLE IX--continued 24-tIUR UPTAKE OF MO AND U BY CMAMIA AND SPIRXGYRA
MG MO/G ALGAE EXPER•IMEUNT PPM. U % REMOVED MG U/G ALGAE PPM ?O 0 RPOVED 2 Spirogyra in Bristol's Control 10.9-1.4 2.5±+0.1 Whole 7.,o±.0 2 8%I 3.1 mg/g 2.7±0.0 13% 0.4 mg/g Part iculate 1.9 -0.5 82% 9.0 mg/g 2.4±0.3 22% 0. 7 mg/g
4A Spirogyra in Shen's Control 21.4±0.8 4.9-0.1 0 Mhole 13.5±0.1 37V% 7.9 mg/g 4.8t1.2 0 Part iculate 6.2±0.1 71% 15.2 rng/g 4.2±+0.6 14% 0.7 mg/g
4B Spirogyra in pond water Control 23.9±1.7 4.5-+0.1 Whole 17.9-±0.3 25% 6.0 Mg/g 4.9+0.2 0 Particulate 5.2-1.2 78% 18.7 mg/g 4.3 0.5 4% 0.2 mg/g
Obtained by dividing the amount in mg of U or Mo removed from solution by the dry weight of algae per flask 41
of uranium did not decrease the percentage uptake, so that the total amount of adsorbed uranium was higher at the higher external concentrations. In the case of molybdenum,
doubling the concentration in solution did not increase the
amount adsorbed, and in fact the percentage uptake was
lower at the higher external concentrations. Disruption of cells increased the amount adsorbed, though on a smaller scale than for uranium.
The material used for the 24-hour studies consisted of living cells, which were left intact in the whole prepara tions and killed only in the omni-mixed preparations. After the 24-hour incubation period, the whole preparations retained their cellular integrity and appeared normal. The particulate suspensions began to lose their chlorophyll pigment after 24 hours. While the solutions from whole preparations were colorless after filtering through 0.45 pm
Millipore filter, the solutions from the disintegrated cells
often were tinted pale brown, which may be an indication
of release of organic acids (Jackson, Jonasson and Skippen,
1978).
F. Long-Term Uptake
The effects of cell growth and subsequent decay on
metal uptake by algae were followed in a series of long
term studies, most of which also involved the interaction
between algal material and pond sediment during decay.
Tables X-XIII give the results from the four long-term 42
experiments.
Experiment I (Table X) involved Spirogyra without added sediment or prolonged decay period. The cultures grew well
for 5 days, after which the algae decayed into fine brown
colored particles. The Bristol's medium used in this test seemed to have precipitated most of the 5 ppm uranium spike, since the day 0 sample taken directly after addition of the uranium contained only 1.2 ppm in the control with out algae. Molybdenum, however, remained in solution. No decrease in either uranium or molybdenum concentrations in the media containing the algae was observed until day 5, which coincided with the onset of decay. After 14 days, the levels of uranium and molybdenum in the containers with algae were reduced about 40% from the day 0 levels.
Experiment II (Table XI) was designed to study the possibility of increased uptake in decaying algae as sug
gested by the results from the first experiment. Cultures of Chara were allowed to grow for 12 days in the light,
after which the containers were placed in the dark and sediment added to two of the algae cultures, in order to
simulate conditions which would prevail in the field as the algae died and sank to the pond floors. The addition of sediment hastened decay of the algae in the dark. Since
none of the containers were bacteria-free, both sediment
containing and sediment-free cultures presumably had increased bacterial activity as algal decay proceeded. 43
TA'RLF. X I_ G-TMI =MPERIMED• I UPTAKE OF URANIUM AND MOLYtUENUM BY SPIROGYRA
DAY PPl U IN SOUT.ION PMT MO IN SOIUTION PIT 1. t0.0o 0 CONTROL 0.1.2±0.5 2+±O. o 4.2 2.1+-0. 8 ALGAE 7.2
1.8-0.0 1 CUTROL 1.7#0.2 4.2 ALGAE 0.9±0.5 2.0±0.3 7.2
3 CONTROL 2.1±0.1 2.0±0.0 4.0 ALGAE 1.6-0.3 2.3±0.3 8.3
1.6-+0.1 5 CONTROL 2.0±0.1 4.3 ALGAE 1.1±0.5 1. 6+0.1 6.9
1.9±0o.0 7 CONTROL 3.5±0.7 4.0 ALGAE 0. 4±0.0 1. 3±0.5 7.5
14 CONTROL 2.8±0.2 2.3-0.0 4.1 ALGAE 0. .. 1.2+0.2 7.8 44
The sediments became black, which is indicative of bacterial sulfate reducing activity. After 23 days in the dark (day
35), the blackening was most prominent around the decaying algae and present otherwise only below the surface in the sediment.
The addition of sediment in control containers increased the uranium in solution but not the molybdenum (Table XI).
Levels of uranium were consistently higher in Chara containers than in either control condition. It is possible that the presence of the algae prevented precipitation of uranium which occured in control containers. After day 35, uranium levels dropped in algae cultures both with and without sediment (Table XI). In Chara only containers, the uranium concentration dropped from 14.5 ppm at day 35 to
4.8 ppm at day 154. The addition of sediment increased the extent of removal of uranium and also hastened the rate of removal, with levels dropping from 11.9 ppm at day 13 to
1.7 ppm at day 154. The cultures of Chara with sediment were the only condition in which molybdenum levels were lowered, with a decrease from 3.0 ppm at day 13 to 1.0 ppm at day 154. The decrease of both uranium and molybdenum in the Chara plus sediment cultures seemed to have reached equilibrium between day 70 and day 154, and no further release of metals was observed.
Experiment III (Table XII) repeated the conditions of
Experiment II but used Shen's medium, which did not precip itate uranium to the degree observed in Bristol's medium. 45
TABLE XI IMNC--TM.IM PDERRIMFN II UPTAKE OF URANIUM AND MOLYJDE=1., BY CT-TARA IN PRESENCE OF SFDIRMJT
PPM IN SOLUTION DAY ChNTROL CHUAA 0 9.4±2.3 10.2±4.1 MOLYBDENUM 2.7±+0.1 2.6±0.1 PH 7.7 7.7
4 URANIUM 1.2±0.7 8.4±0.9 MOLYEDENM., 2. 7±. 1 2.6-+0.1 PH 7.1 7.8
8 URANIUM 1.1±0.5 15.9±7.1 MOLYBDEUM 2.7±0.2 2.8±0.2 PH 7.1 7.7
12 URANIUM 2.1±0.8 14.4±1.5 MOLYBDENU. 2.7±0.1 2.88±0.1 PH 7.0 7.9 SEDIMIENT ADDED SEDI•MNT ADDED 13 URANIUM 2.o5±0. 1.0-0.1 13.-2±0.4 11.9-0.3 MOLYBDENDU 2.66±0.0 2.8±0.1 2.8±0.1 3.0-0.0 PH 7.0 7.6 7.6 7.6
21 URANIUI 0.6+-0.2 2.4±1.2 13. 1±1.2 6.7±-0.7 MOLYBDEIUM 2.8±0.0 3.5±0.0 2.7±0.0 2.7±0.0 PIT 7.0 7.8 7.5 7.5
335 URANIU.M 0.9±0.3 ..6±1. 3 14.5±1.8 4.0±0.5 MOLYBDENUM 2.8±0.2 2.9±0.1 2.8±0.1 1.9±0.0 PH 6.9 8.0 7.4 7.7 46
TABLE XI--cont inued LONG-TFMI =FRPERIMENT II UPTAKE OF URANIUl AND MOLYEDFNUI BY CHARA IN PRESENCE OF SFDI;IENI'
PPM IN SOLUTION DAY CONTROL CHAPA S1RTMFý7 ADDED SfD I•MFT ADDMD 1.1±0o.2 70 URANIUM 0.2±+0.0 8.3±22. 1.9±0.4 MOLYBDENUM 3.4±0.1 2.9±0.2 3.1-10.2 0. 8-0.0 PH 7.1 8.2 7.8 S.2
154 URANILM 0.1±0.0 3.88±1.8 4.8 1.710.0 MOLYDENRM 3.2±0.4 3.2±0.1 3.2±0.1 1.0±0.0 PH 7.1 8.1 8.1 8.1 47
The Spirogyra cultures contained some Oscillatoria, which lysed within 1-4 days, giving the medium a faint brown color. The Chara cultures showed new growth throughout the 13-day light period, while the Spirogyra cultures were decaying after 13 days. The Spirogyra containers had developed a surface growth of blue-green algae, predominately
Lyngbya, by the end of the light period. By day 21, the control sediments without algae had a light, oxidized surface layer on the sediment, while the sediments with algae remained black throughout. After 21 days in the
Spirogyra cultures and after 49 days in the Chara cultures, the blackening of the sediment surface and the presence of intact algae cells were no longer evident.
Removal of uranium was most pronounced in the Spirogyra cultures (Table XII). While the presence of sediment did not enhance the removal of uranium from solution in compar ison to the sediment control, the algae increased the sediment's ability to retain uranium. At day 166, 2.1 ppm uranium remained in solution in the Spirogyra-sediment cultures and 3.8 ppm in the sediment control.
In the Chara cultures, growing algae had no effect on uranium removal, and some release of uranium from Chara
seemed to have occured when the containers were transferred
to the dark (Table XII, days 0-14). However, as Chara
decayed in the presence of sediment, levels of uranium fell
from 8.0 ppm at day 14 to 2.0 ppm at day 49. Lessened TABLE XII LONG-TERM IMfERIME=T1' III UP:AIK OF URANIUM AND MOLY•UDEUM BY CIIARA AND SPIRGX3YRA IN PRESECE OF SEDIhENT
PPM IN SOIYFION DAY CONTROL CHARA SPIROGYRA 0 URANIUM 5.8±1.9 5.8±0.8 5.3±2.1 0,!OLYJ3DNL'M, 2.2±0.3 1. 9±0.2 2.3±0.2 PH 7.4 7.8 7.8
3 ,PANIUM 5.1±2.2 6. 7o.9 4.2±0.7 M.!OLYBDENU• 2.2±0.2 2.3±0.2 2.0±0.7 PH 7.4 7.7 7.8
7 URANIUI 3.3±0.6 5.3 0.9 3.5±1.2 MOLYBDENUM 2.2±0.2 2.2±0.2 1.9+0.3 PH 7.4 8.0 7.9
13 URANIUM 3.0±0.3 5.5±1.2 2.1±0.3 MOLYBDENUM 2.9±0.6 2.2±0.2 1. 9±-0.4 PH 7.4 8.0 7.7 SEDIMET ADDED SFDIM•NT ADDED SEDIMENT ADD]I) 14 URANIUM 4.3±0.5 63.0-1.5 7.8±0.1 8.1±1.0 3.8±0.8 3.5±+0.8 MOLYBD!IUM 1. 9±0.1 2.2+0.0 2.0±0.1 2.4±0.0 1.4+0.1 1.9±0. 1 PH 7.4 7.7 7.8 7.9 7.5 7.7 TABLE, XI I--continued LONG-TERM XPF!IMENT III UPTAKE OF URANIUM AND MOLYBDENUM BY CHARA AND SPIROGYRA IN PRESENCLE OF SFDIMENT
PPM IN 9DLUTION DAY CONTROL ClARA SPIROGYRA SFDIMENT ADDED SEDIM.1r ADDED SEDIMENT ADDED 21 URANIUM 2.7±0 .5 4.6±0.(6 6.3±1.2 3.2±0.0 2.0±0.1 1.2±0.2 MOLYTBEWNU 2.4±0.0 2.0±0.5 2.5±0.0 2.3±0.0 2.5+o0. 0 1.0±0.1 PH 7.4 8.1 7.6 7.8 7.7 7.6
49 URANIUM 3.7±0.1 5.2±0.0 6.5±0.3 2.0±0.0 1.7±0.6 1.5-0.2 MOLYEDENUM 2.3±0.0 2.7±0.1 2.9±0. 6 1.8±0.3 1.6±0.1 1.5±0.3 PH 7.4 7.7 7.8 7.8 7.8 7.6
80 UR-ANIUM 4.8±1.1 5.5-0.4 7.2±0.5 4.2±0.9 2.5±0.3 4.3±1.1 MOLYBDENUM 2.1±0.0 2.8±0.1 2.3±0.1 1.81+0.5 2.2±+0.3 2.7±0.2 PH 7.6 7.6 7.6 7.6 6.0 7.6
166 LlRANIUM 4.8±0.7 3.8-0O.5 5.5±0.2 3.-8±1.1 0.6±0.0 2.1±0.5 MOLYBDENUM 2.9±0.3 2.3±0.2 3.8±+0.1 4.1±1.4 3.2±0.2 4.7±0.3 PH 7.4 7.3 5.9 7.7 50
decay of the algae after day 49 allowed re-establishment
of an oxidizing zone at the sediment surface, which coincided with partial remobilization of uranium. The similarity of
day 80 and day 166 values, 4.2 ppm and 3.8 ppm respectively,
suggests that an equilibrium had been established in the
system.
No cultures in Experiment III showed irreversible
removal of molybdenum, although Spirogyra cultures reduced
molybdenum substantially by day 21, from 2.2 ppm to 1.4 ppm.
However, molybdenum levels returned to original levels in
solution in all cultures and were, in fact, higher than the
initial values in the algae and sediment cultures. Experiment IV (Table XIII) was intended to magnify the
effect of decaying algal material on sediment retention of
uranium and molybdenum. The amounts of sediment and algal
material were increased (Table IV), and the algal material,
dried Chara, was dead at the start of the experiment. By
day 16, bacterially mediated decay was extremely vigorous
in the algae cultures, which were heavily overgrown with a
surface layer of bacteria. The algae plus sediment cultures
were blackened throughout the solution and sediment, while
the sediment alone cultures were blackened only beneath
the surface of the sediment. High bacterial activity was
evident by visible inspection even after 47 days. However,
after day 74 (Table XIV), the algae cultures had lost the
blackened appearance, and the surface masses of bacteria 51
TABLE XIII LONG-TFRM =ERIMENT IV UPTAKE OF URANIUM AND MOLYNUMBDE BY CTIARA AND SEDIMENT IN DARK-MAINTAINED CULTURES
DAY Pr4' U PH 2 SEDIN= 24.3±0.1 6.3±0.5 ALGAE 15.3±2.5 7.3±0.6 SEDI.ENT+ALGAE 1.2. o..'5 4.2±0.2
16 SEDIMENT 8.6±0.4 4.2±0.5 7.61 AIJ3AE 7.2±0.3 2.6±0.2 7.31 SED.INrT+ALGAE 1.0±0.2 0.4±0.4 7.23
47 SEDIM•NT 2.6±0.3 2.7±-0.4 7.75 ALGAE 4.1±1.1 3.1±0.1 7.50 SEDIMENT+AIfLAE 0.3 0.06-0.05 7.80
SEDIMENT 108 1.2-0.3 1.4±+0.4 7.62 ALGAE 3.9±1.2 3.0±0.0 7.72 SEDIMFNT+ALGAE 1.0±0.4 0. 03±0. 01 8.02
190 SED IMET 0.7-±0.0 1.7±-o.1 7.70 ALGAE 1.1 3.2-+0.3 7.79 SED IMENTM+ALGAE 1. 9±0. -I 1.0--0.0 7.90
All cultures wore spikod at day 0 with 20 ppm U and 5 ppm Mlo 52
were absent. In the sediment plus algae cultures, the algae and sediment surfaces were still blackened, but an oxidized
layer had formed in the sediment alone. The Chara cells
remained partially intact even at the end of the test,
though most of the algal material had disintegrated into particles. Table XIV gives Eh values in the cultures for
days 74, 108 and 190. Both water and sediment were reduc
ing at day 74, but by day 108 only the sediment remained reducing.
The percentage removal-of 20 ppm uranium and the 5 ppm molybdenum spikes which were added to all containers is given in Table XV. Initial release of uranium from sediment and from algae containers was seen at day 2. Sediment alone removed more uranium (941) and molybdenum (72%) than algae alone (80% for uranium and 40% for molybdenum), but the combination of sediment plus algae increased removal of uranium to 95% and of molybdenum to 991. Removal of both metals was essentially complete after 16 days in the
algae plus sediment cultures, but some release of both occurred by day 190. This release was not observed in
either the sediment alone or the algae alone. 53
TABLE XIV EH NMEASUIRTiNS FOR IONG--ITR. MOM=FIMIl IV: CHARA AND SEDIMENT IN DARK-MAINTAINJ2D CULTLRES
DAY SEDIMENT AI_.AE SEDIDMT+ALGA3A water sediment water water sediment 74 -204±5 -502±46 -415±5 -302±2 -515±25
108 +150±4 -339±(3 +107±2 +88±1 -392±23
190 +68±1 -382±80 +70±0 +50±25 -393±105
TABLE XV • I.ONG-TF.-T EXPm.PTNT IV PERCMNTAGF, OF URANIUIM AND MOLYTnF•UJ RPInOVFD BY CHARA AND SEDIHME IN DARK-MAII\TAIN1D CULTURES
DAY SEDIM•ET ALGAE SEDDIMEN+ALOAE U MO U MO U MO 2 +231-, +26% 23ýT +46% 36% 16%
16 57%, 16% 64% 48W. 95% 99C71
47 87% 461", 79M 38% 98. 991..
108 94% 72" 80% 40% 9,5o,,.
190 96. 36%9 94% 36% 90% 80%
Plus sign indicates a percentage increase over the amount of spike added at day 0; values otherwise represent a percent age decrease. 54
IV. DISCUSSION
The marked differences in accumulation of uranium and molybdenum by the different algae in the pond system may be due to the different chemistry of the two metals, but the roles of the metals in the algal life cycle may also play a part in determining their accumulation.
Molybdenum is probably present in the pond water as 2 the anion MoO 4 (Bloomfield and Kelso, 1973), and its accumulation by direct adsorption onto negatively charged algal cell surfaces seems unlikely. Bloomfield and Kelso
(1973), however, do point to the suggestion of Szalay
(1969, Arkiv. Mineralogi Geologi 5: 23-36) that molybdenum is reduced by organic matter to exchangeable cationic forms, so that adsorption of molybdenum may occur in algae.
Molybdenum can be an essential trace element for green algae, and its requirement for those blue-green algae which fix nitrogen has been demonstrated (O'Kelly, 1974). It is possible that cells requiring molybdenum would have mechanisms to regulate levels of the metal in the cell interior. Chara in particular shows little variation in molybdenum levels. While a regulatory system for molybdenum has not been demonstrated in Chara, Robinson (1969) has found active uptake of sulfate ions into the vacuole of
Chara australis by a pump across the plasmalemma, which might provide a pathway for uptake of MoO4 2 as well. In 55
the field-collected algae, levels of molybdenum varied
10-fold over different collections, which may be due to adsorption onto cells in addition to uptake into the cells.
The ability of Spirogyra to adsorb molybdenum on a short term basis was indicated by the 24-hour uptake experiments, where an increase in surface area led to an increase in removal of molybdenum from solution. However, the differ ent levels in the collections may also be due to varying populations of algae over the year, especially since the winter collection, which showed higher molybdenum content, also contained a higher proportion of the blue-green
Oscillatoria.
In contrast to molybdenum, uranium is generally considered an abiological element (Taylor, 1979), and its uptake is more likely to be governed by physical-chemical
factors than by active cellular processes. The uranyl ion has an extremely high affinity for organic material
(Jackson, Jonasson and Skippen, 1978), and adsorption
onto the negatively charged algal cell surfaces probably
accounts for the high levels of uranium in the pond algae.
Chara shows a lower affinity for uranium than the filamen
tous algae. In a study of radionuclide accumulation by
Chara species, Marchyulenine (1978) felt that the presence
of high levels of calcium carbonate compounds in the cell wall reduced the number of cation exchange sites in Chara
compared to other algae. Sikes (1978) noted that in another 56
calcium-accumulating alga, Cladophora, calcium carbonate
increased with the age of the cells, and this calcium was
not subject to appreciable ion exchange.
The seasonal variation in both uranium and molybdenum
levels in the filamentous algae suggests that adsorptive
processes are important in the accumulation of the metals
in the algae cells, since extent of adsorption depends not
only on the amount of metal available per unit of surface
area but also on the length of exposure of the surface to
the metal. Thus, longer-lived cells would be exposed to
more metals than short-lived cells. The September
collections represent quickly growing populations, while
the January collections were taken from sparser populations
which, due to the slower growth rate in winter, had a
higher turnover time than the summer populations. April may
represent a transition from chiefly perennial populations
to annual summer blooms of algae. Knauer and Martin (1973)
found that levels of heavy metals in marine phytoplankton
were low during periods of algal blooms due to dilution of
the amount of metal available per unit mass of phytoplankton.
While the algae ponds in this study did support denser
populations in the summer, the increase in cell number probably was not great enough to alter the ratio of metal
to algae. More important is the increased length of expos
ure time for the winter populations. Hodge, Koide and
Goldberg (1979) found that marine macroalgae adsorbed 57
uranium, plutonium, and polonium in direct relationship to exposure time. The longer exposure could also lead to exchange absorption into the cell interior, as described in the long-term exposure of Cladophora to lead (Gale and
Wixson, 1979).
The results of the 24-hour uptake experiments support the field evidence for different interactions between the metals and the different algae. Short-term uptake was not observed in Chara, which in fact accumulated both metals at much lower levels than the filamentous algae in the field.
In the Spirogyra 24-hour test, the cell material showed a
limited capacity to adsorb molybdenum, while uranium uptake
increased in higher external concentrations. The disruption of the cells to form particulate suspensions evidently
increased surface area for binding, although release of
cellular constituents, which may form insoluble complexes, might also have been a factor (Ferguson and Bubela, 1974).
Horikoshi et al. (1979) also observed an increase in
adsorption of uranium in disrupted cells. In an 8 ppm
solution of uranium, adsorption increased from 15,600 ppm
in living Chlorella to 67,200 ppm in heat-killed cells.
The long-Ierm 1.aboratory studies indicate that the pond
algae, in the form of particulate, decaying material, can
be instrumental in removing metals from solution. However,
the patterns of retention and release of uranium and molyb
denum as the algae decayed in the presence of sediment
indicate that maintenance of reducing conditions in the 38
sediment or in the algal cultures is critical to the sequestering of the metals. In the model systems, the period of algal decomposition was associated with increased bacterial activity and an enhancement of reducing conditions in the sediment-containing cultures. The algae cultures without sediment probably decomposed mainly in aerobic conditions, except in Experiment IV, where the high propor tion of algal material led to reducing conditions in the first two months of incubation. When sulfate levels are high, as they are in the pond water, anaerobic decomposition of algae is accompanied by sulfate reduction even in the absence of sediment (Foree and McCarty, 1970). Decomposi tion was associated in the model systems with removal of molybdenum and uranium from solution, but the period following decompostion was characterized by the partial to complete release of the metals back into solution. The release of molybdenum was surprising, since sulfides which are formed during sulfate reduction can form highly insol uble molybdenum disulfide. It is possible that molybdenum was precipitated in the reduced Mo(IV) state but did not form the sulfide mineral, so that release occurred as molybdenum was re-oxidized. IHowever, molybdenum showed little or no release in Experiment IV, although oxidizing conditions were established in the water compartment and at the sediment surface for at least the last 82 days of the experiment. 59
In most cases, the addition of algae to sediment
increased the sediment's ability to retain uranium.
Reduction of uranium to the insoluble U(IV) ion may have
contributed to the removal, with adsorption onto the organic
material another possibility. Taylor (1979) has pointed
out that in sediments, the production of hydrogen sulfide
by sulfate-reducing bacteria may increase the adsorption of
uranyl ion onto organic material present in the sediment,
since iron and other sulfide-forming metals could be
desorbed from the organic material to increase adsorption sites for uranyl ion.
The implication from the laboratory studies is that, while the algae are instrumental in removing metals from solution, the process is reversible unless the system contains substantial organic material. Also, while organic material accelerates the rate of removal and sometimes the extent of removal from water, retention of the metals in sediments is also reversible unless the system contains a high volume of sediment. These conclusions point to major problems in improving the existing pond system in respect to removal of uranium and molybdenum.
In the present system, uranium levels are reduced significantly between the initial influent from the mines and the final effluent, but the removal is evidently effected almost entirely by the ion exchange system in section 35, and possibly by precipitation in the large 60
section 36 settling pond. Since the rate of flow from both sections 35 and 36 is about the same (2x106 gallons day- ), the average input of uranium into the system as a whole
can be calculated as the average of the two sections, which
is 4.0 ppm uranium (see Table I for pond water values).
The reduction to 0.7 ppm in the final effluent is signifi cant at the .01 level. However, as can be inferred from the similar averages of uranium concentrations in the three algae ponds, there is no significant reduction of uranium in solution in any of the algae ponds or indeed between the influent to the algae ponds and the final effluent.
The case of molybdenum is similar. There is no significant reduction of molybdenum in solution throughout the system; the values in the algae ponds are lower than in section 35 because of mixing with the waters from section 36, which have very low levels of dissolved molybdenum. Although the sediments concentrate both metals susbstantially over concentrations in water, the amount of metals removed is not substantial enough to be reflected in the water concentrations.
Part of the problem in seeing statistically significant trends in the data is due to wide fluctuations over the samples values, as indicated by the relatively high standard deviations in Table I. More data might overcome part cf this problem, if in fact there are trends which are being masked by the present sampling regime. However, k 1
it is important to remember the volume of water which passes through the system yearly. At a combined inflow of
4x106 gallons a day, or 15x106 liters a day, the pond system receives 5.5x10 9 1iters every year. Based on the uranium and molybdenum values in the influent to the algae ponds of
0.9 ppm for each metal, this volume amounts to 4.95x109 mg
(4.95 metric tons) each of molybdenum and uranium entering the algae ponds yearly. The filamentous algae present in the ponds now accumulate an average of 2000 ppm uranium and
130 ppm molybdenum on a dry weight basis. Given this level of removal, it would take 4.95x109 mg U x (103 g algae/2000 mg U) = 2.5x109 g dry weight algae produced per year to remove the uranium entering the algae ponds. If the ponds were fertilized to increase productivity, could this amount of algae be grown in the existing pond system? Wetzel
(1966) found that a hypereutrophic lake produced 570 g C m-2 yr -1. Using a common value found for carbon content in algae of 40% of dry weight (Ferguson and Bubela, 1974), -2 algae of 1425 g m 570 g C corresponds to a dry weight of -1 9 -1I yr In order to grow 2.5x10 g algae yr , the amount needed to accumulate the influent uranium, the pond system
would have to be expanded to 2.5x109g x (m 2/1425 g) 6 2 1.75xi0 m2, or 17.5 hectares. The ponds now cover 0.15 hectares. Obviously, relying on algal removal alone will
not answer the problem of improving the pond system.
Increasing pond productivity may improve removal 62
indirectly, as suggested by the long-term laboratory studies. The ponds are fairly unproductive now, which is
typical of the early to middle stages of succession in
Chara ponds, where phosphate levels are quite low (Crawford,
1979). Later stages of Chara succession are characterized
by overgrowth of planktonic algae and higher plants, which
increase pond productivity. Additions of phosphate and
nitrogen-containing fertilizers would accelerate the replace ment of Chara with more productive annual green and blue
green planktonic algae. Promoting eutrophication of the
pond system would increase the amount of organic substrate
available for sulfate-reducing bacteria and would promote
reducing conditions in the pond sediments. Laboratory
experimentation has indicated that enhancement of growth
of Desulfovibrio and Desulfotomaculum and thereby of
sulfate reduction by these bacteria in sediments resulted
in removal of uranium, molybdenum and selenium from
solution (Brierley and Brierley, 1980). It is possible
that, while the algae cannot function alone to remove
excess trace contaminants, the effect of increased algal
productivity on water and sediment chemistry might be
significant. 63
APPENDIX A ANALYTICAL PRWCEDURLES
A. The following procedure for uranium analysis is taken from the nethod of Johnson and Florence (1971).
Uranyl ion can be separated from contaminants by solvent extraction using trioctylphosphine oxide in cyclohexane. A yellow colored conplex of uranium (VI) dibenzoylmethane form instantly by introducing an aloquot of the extract into a pyridine-ethanol solution of dibenzoylmethane. The analysis has a sensitivity of 0.05 ppm uranium.
Reagents
1M hydroxylamine sulfate: Dissolve 164.14 g of hydroxylamine sulfate in 1 liter of distilled water.
D1I potassium flouride: Dissolve 58.1 g of KF in I liter of distilled water.
0.051M trioctylphosphine Dissolve 19.3 g of trioctylphosphine oxide in cyclohexane: oxide in 1 liter of cyclohexane.
507o pyridine in ethanol: Mix 1:1 pyridine in ethanol.
1% mixed color reagent: Dissolve 10 g of dibenzoylmethane in 1 liter of absolute ethanol. uranium standard: Dissolve 1.0000 g of pure U3 0 in a minimum amount of nitric acid and dilute to 1 liter with distilled water. working standard: Dilute 1 ml of 1000 ppm standard to 100 ml with distilled water. Make fresh daily.
Procedure
1. Prepare blank and standards of 0.1, 0.2, 0.3 and 0.5 ppm 113O8 using the working standard of 10 "pm.
2. Dilute blank, standards and sarples to 100 ml in 250 ml separa tory funnels. 64
3. Add 2 ml of concentrated IMO3.
4. Add 1 ml of hydroxylamine sulfate solution to reduce iron.
5. Add 1 ml of KF solution to conrplex molybdenum, zirconium and titanium.
6. Add 5 ml of TOPO in cyclohexane.
7. Stopper the funnel, shake gently, relieve internal pressure, and shake vigorously for 2 minutes.
8. Allow sample to stand until phases separate, about 5 minutes.
9. Drain off and discard bottom aqueous layer.
10. With a dry pipet, remove 2.0 ml of the organic phase and transfer to dry 10 ml volumetric flask.
11. Add approximately 1 ml of the 5MY. pyridine-ethanol solution to the sample until a clear solution is obtained.
12. Add 2.0 ml of the mixed color reagent.
13. Bring to volume with the 50% pyridine-ethanol solution and mix well.
14. Allow the sample to stand for at least 5 minutes for color development.
15. Set spectrophotometer to zero absorbance with the blank at 405 nrm. Read absorbance of standards and samples.
16. Prepare standard curve and calculate the concentrations of the samples.
B. The following molybdenum analysis is taken from Meglen and Glaze
(1973).
Molybdenum is analysized colorimetrically as the thiocyanate
molybdenum complex. ITe analysis has a sensitivity of about 5 ppb
molybdenum. C)5
Reagents
1% ferrous annonium Dissolve 1 g Fe(N7 4 SO4 )2 in 100 ml dis sulfate: tilled water with 1 mil concentrated sulfuric acid.
10% potassium thiocyanate: Dissolve 5 g KSCN in 45 ml of distilled water. Make fresh daily.
10% stannous chloride: Dissolve 100 g SnCI2 in 125 ml concentrat ed nitric acid, heat to dissolve. Add slowly to 900 ml distilled water. Store in container with piece of pure tin metal.
Procedure
I. Prepare blank and standards of 50, 100, 300 and 500 ppb Mo in 250 ml separatory funnels.
2. Dilute blank, standards and samples to 100 ml.
3. Add 2 ml of concentrated HCI.
4. Add approximately 0.2 g sodium tartarate to co, plex tungsten.
5. Add 0.5 ml of 1% ferrous annonium sulfate.
6. Add 3.0 ml of KSCN solution.
7. Allow sample to stand 15 minutes to develop pink to red color.
8. Add 9.0 ml of 10% stannous chloride solution.
9. Allow sample to stand 15 minutes. Pale arrber thiocyanate molybdenum complex will form.
10. Add 10.0 ml of iso-amyl alcohol. Shake for I minute.
11. Allow phases to separate, about 15 minutes. Drain off and discard bottom aqueous layer.
12. Using'dry pipet, remove aliquot of organic phase and transfer to spectrophotoupeter tube.
13. Set spectrophotometer to zero absorbance with the blank at 465 nm. Read absorbance of standards and samples.
14. Prepare standard curve and calculate the concentrations of the samples. I .6
APPENDIX B
STATISTICAL TEST VALUES
One-way analyses of variance were applied to data collected over the study period in order to determine whether differences between ponds or between algae collections were statistically significant.
In tests 1-7 in the table below, different pond sites were compared.
Thus, the classes are collection sites, as described in Table I, and the values within a class are the monthly sanple levels. In tests 8 and 9, the metal levels in the three algae collections were compared, with the levels from different sample sites (described in
Table VII) constituting the values within a class. Tests were considered significant if the calculated F value was greater than the F value at P=-0.01 (taken from Snedecor and Cochran, 1967).
DEGREES OF FREEDOM F VALUE CALCULATED DESCRIPTION OF TEST Between Within P8-O. 01 F VALUE Classes Classes 1 16 8.53 10.00 Uranium: combined minewaters section 35 and 96 compared to effluent algae pond 3
1 16 8.53 10.46 Uranium: combined minewaters section 35 and 'IScompared to influent algae pond 1
3 20 4.94 0.93 Uranium: comparison of ponds in section '5 azwong themselves
1 1-2 9.33 3..36 Uranium: minewater section 36 compared to sec. 36 pond 3-2 effluent
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Sikes, S. C. 1978. Calcification and cation sorption of Cladophora glomerata (Chlorophyta). J. Phycol. 14: 325-329. 71
Johnson, D. A. and T. M. Florence. 1971. Spectrophoto metric determination of uranium (VI) with 2-(5-bromo-2 p ridylazo)-5-diethylaminophenol. Anal. Chim. Acta 53: /3-•-79.
y D. P., P. R. Norris and C. L. Brierley. 1979. Microbiological methods for the extraction and recovery ,of metals. In: A. T. Bull, D. C. Ellwood and C. Ratledge (eds.), Microbial Technology: Current State Fu re Prospects, Cambridge Univ. Press, London, pp. 263 3 8.
aue G. A. and J. H. Martin. 1973. Seasonal varia tions of cadmium, copper, manganese, lead and zinc in X/water and phytoplankton in Monterey Bay, California. Limnol. Oceanog. 18: 597-604.
SLaube, V., S. Ramamoorthy and D. J. Kushner. 1979. Mobilization and accumulation of sediment bound heavy metals by algae. Bull. Environ. Contam. Toxicol. 21: 763-770.
Leland, H. V., S. N. Luoma, J. F. Elder and D. J. Wilkes. 1978. Heavy metals and related trace elements. J. Water P. C. Fed. 50: 1469-1514.
Mang'/S" and H. W. Tromballa. 1978. Aufnahme von Cadmium i•rch Chlorella fusca. Z. Pflanzenphys. 90: 293-302.
yulenin , E.-D. P. 1978. Exchange of certain radio clides between the environment and freshwater algae. Soviet J. Ecol. 9: 163-165.
Matzku, S. and E. Broda. 1970. Die Zinkaufnahme in das Innere von Chlorella. Planta 92: 29-40.
McDuffie, B., I. El-Barbary, G. J. Ilollod and R. D. Tiberio. 1976. Trace metals in rivers - speciation, transport, and role of sediments. In: D. D. Hemphill (ed.), Trace Substances in Environmental Health-X, Univ. Missouri, Columbia, pp. 85-95. 72
Meglen, R. R. and M. L. Glaze. 1973. Analytical methods. Report to the Molybdenum Project, No. MoP/g-73/MW, University of Colorado, Boulder.
Morris, A. W. 1971. Trace metal variations in sea water of the Menai Straits caused by a bloom of Phaeocystis. Nature 233: 427-428.
O'Kelly, J. C. 1974. Inorganic nutrients. In: W. D. P. Stewart (ed.), Algal Physiology and Biochemis try, Univ. Calif. Press, Berkeley, pp. 610-635.
Prescott, G. W. 1954. How to Know the Freshwater Algae. 2nd edition, W. C. Brown Co., Dubuque, 348 pp.
Ray, S. and W. White. 1976. Selected aquatic plants as indicator species for heavy metal pollution. J. Environ. Sci. Health A-I1: 717-725.
Robi son, J. B. 1969. Sulphate influx in characean cells. I. General characteristics. J. Exp. Bot. 20: 201-211.
e:ige, U. and P. Edwards. 1977. Correlation coeffi '.ients and concentration factors of copper and lead in Is'e vater and benthic algae. Marine Poll. Bull. .17:
-19. eliger and P. Edwards. 1979. Fate of biologically cumulated copper in growing and decomposing thalli of vatbenthic red marine algae. J. Mar. Biol. Ass. U. K. 59: 2277-238.
Shen, E. Y. F. 1971. Cultivation of Chara in defined medium. In: B. C. Parker and R. M. Brown, Jr. (eds.), Contributions in Phycology, Allen Press, Lawrence, Kansas, pp. 153-162.
Sikes, S. C. 1978. Calcification and cation sorption of Cladophora glomerata (Chlorophyta). J. Phycol. 14: 325-329. 73
Smith, G. M. 1950. The Fresh-Water Algae of the United States. 2nd edition. McGraw-Hill, New York, 719 pp.
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Institute by the following committee:
L,9
Date PREPRIFiT 16TENDED ABSTRACT' Presented Bfore the Dilvlsion of EnvironLental Chemistry American Chemical Society Denver. Colorado April 5-10. 1957
RADLOWUCLIDC IN NPIADA IEST SITS CAO•CNDW<: "TRANSPORT BY CLAY COUAIDS 1 2 A. W. Duddeiler and J. R. Hunt 1 Lawrence Livermore National taboratory P. 0. Box 808, L-233 Livermore. California 94550
Department of Civil vngineerirn2 University of CalifornLa Berkeley, California 9470%
the Cheshire (U2On) event was a nuclear detonation fired February it. 1976, at the Nevada Test Site (NYS). It was detonated in a fractured ryholitie Lava format ion 1167 m below the surface and 537 a below the water table. Yield was in the 200 to 500 kiloton range. A slant-drlled re-entry hole was cased and perforiated within the cavity. Cavity water was Intermittently ptuped and ammpled between September 1983 and May 1985. Thereafter. the well was plugged at 860 w and pumping was' resued from new perforations In the 763-858 a depth range. Because or the slant of the hole the new location was estimated to be approximately 300 a above the cavity and displaced approximately 100 a hydrologically down-gradient. Because a number of strongly sorbing or irnoluble ralonuuolldee were observed In the "dasaolved" phase (0.45 um filtered) of early cavity samples, Lawrence Livermre National Laboratory Initiated a Series of filtration and ultrafiltration experiments using large-volume samples. Filtratea and ultrafiltrates were dried, weighed, gamm counted and further characterized; filters were g•aa counted. Table 1 presents the dLetributLo or' masses between the dissolved phase and various particulate size ranges based on weight relationships.
Table I: Dissolved and Colloidal Material in Cheshire Samplesa
Colloid Est. Colloid Sample T70 (mg/L)b size range (m) Mass Con. (mg/Ll B-I (cavity) 256 >0.006 55 B-2 (cavity) (256) (0.45 - 0.006) (63) 8-3 (cavity) (263) (0.2 - 0.003) (35) (0.2 - 0.05) (25) 0-4 (cavity) 263 0.05 - 0.003 10.1 0-5 (external) 216 0.05 - 0.003 4.6 B-6 (external) 218 0.05 - 0.003 4.3
otes-: avalues in parentheses calculated for nos-ultratiltered samples by assuming jDS f'or other samples applied. calculated from weight of salts recovered fro* dried ultrafiltrate.
X-ray dit'fraction studies of iltrafiltrate and retentate solids showed that the ultraf I tretes were composed or evaporite salts (halite. trona, arapnlte), but that the retentatee were dominated by quartz and (Ca, K) reldspers. The equilibrium geocbemical model code 3Q6 was run for a variety of scenarios using both laboratory and field pH and temperature observations and water chemistry determined by analysis
"534 or 0.S5 - filtered aaples. All runs predicted the Presence of a solid phase dOmlnatee py quarts (5H - 60 g/OL) and oontaLln•I 1-10 Mg/[. muscovte and phenglta (fiele temperature and pH) or kaolinite and pH). nontroLte-ca (iab temperature and Although ohemical equllibrium Is conditions, not necesarily a valid guide to both the mass rangee and actual the types of mlinerals predicted are qualitative agreement with the observatlons. In X-ray fluorescence results for the Ultrarilt•-te and retentate free are presented In Table 2. Since sample D-4 the actual mass in the 0.05-0.003 3im range Is 3.04 of the TDO, the elements particle size K. Fe, 14n, Rb, Pb. Cs, On. Le quite concentrated in the colloidaL and Ce are phase. The potasesim effect Is ascribable the potasslam-bearing *lay minerals to In the solids. while the other amon those whose sorption elements are or solublity characteristics cake them candidates for particulate association. logical
Table li: X-ray Fluorescence Analysis, Sample S-4 Cal*. Colloidal Element Salts (ppm) Retentates P lEa t.(05-O. Val
I 1800 25400 Ca 0.356 2900 2200 0.028 Mn 96 540 0.180 Fe 318 547TO Ut 0.403 32 20 Cu 159 0.023 280 0.064 Zn 1980 610 0.011 M9 22 0.016 ri0 19 0.004 Rb 19 190 MO 0.261 11 6 Pb 0.004 35 320 C 0.263 <5 14 >0.TO8 B: 25 203 0.21] La <5 19 >0.129 Ce <5 SM >0.397 Table 3 presents data on the traction of the total actility passing ftlter (or ultraliter) for those the final nuolides observed in all or most of Total concentrationa of the more the samples. soluble nuclidea (Sb, Ca) w•ee only (or less) lower at the external a factor of two location tMan In the cavity, btt total concentrations of Mn and Co radlonuclides were reduced by factors concentrations of the lanthanide of 1-5 or more and nuolIdes were more than an order lower outside of the cavity. of sagnittd•e Table 3 shows very signirtcant difrerences between the ultrariltered conventionaLly filtered cavity and the samples, and somewhat, subtler differences ultratiltered cavity and external between the samples. le also note that the different isotopes of the behaviors of same element are generally quite similar. We can aummarize the following major conclustons fero these obseryationaz 1. Essenttally all of the transition element and lanthanide nuclides are associated with particles, observed the bulk or which are not filterable standard filtration techniques. by
2. The pre-sence of these nuclides outside of the cavity, albeit at concentcrations greatly reduced relative to more conservative strongly suggeats tracers, that they are moving by particle transport.
535 3. fhe available chealtcav aineralogleal and geoohemleal data strongly sugg•et that the partiolea involved are ColLoidal clays whLch serve as a substrate for the treasport of sorbed nuolides.
Table LII: Fraction of Activity Pas-ing Final Filter Sample: 91 32 63 54 95 (Cavity) (Cavity) (Cavity) (Cavity) PIL]ter a- (External) (00): 0.006 0.5 0.2 0.003 0.003 0.003
Nuolide
2 2 m_ 0.95 0.98 0.97 0.89 0.95 -1.00 0.33 0.76 0.97 0.31 a0.73 0.83
1 Work per'formed under the auspices of the U.S. Department of Energy by the Larence IC Livermz•Ore National Laboratory under czntract No. t n-705-E ehG-h8.
w. I
536 0
BIODEGRADATION OF DRILLIN FLUIDS: -: . " -•-•" EFFECTS ON WATER GMUSTRY "AND ACTINIDE SORPTION
by
Larry E. Hersman
ABSTACT.
Several million gallons of drilling fluids have been introduced into, or Inmediately adjacent to, the candidate high-level nuclear waste repository at Yucca Mountain, Nevada. Studies conducted by the Nevada Nuclear Waste Storage Investigations (NN1SI) Project demonstrate that these drilling fluids are biodegradable by a variety of micro organisms. Further studies demonstrfte that one species of "bacteria is able to strongly sorb Pu".
Several million gallons of drilling fluids were used during characterization .of the geology and hydrology of the site for a candidate high-level'nUclear waste repository at Yucca Mountain, Nevada. T1his activity was performid by engineers involved in the Nevada Nuclear Waste Storage Investigations(.N{WSI) Project. The drilling logs from wells on the site indicate that a significant amount of the fluids was lost in the Topopah Spring Member, .the geologic location of the candidate repository. The Topopah Spring Member has been characterized as a weldeq tuff with low permeability and high fracture content. Montazer and Wilson have suggested that in this member lateral movement may exceed downward rates, a statement substantiated by the recent discovery of drilling fluids in the USW UZ-l hole (air drilled without fluids)., Interestingly, UZ-i is located several hundred meters upgradient'.framuthe closest fluid-drilled holes (USW G-l and H-l) and its drilling was limited to the unsaturated zone, at a depth approximately equal to that of the repository. In addition to those drilling fluids, contractors plan to introduce several other forms of organic materials into the block candidate site. During the construction of the Exploratory Shaft, items such as diesel fuels and exhausts, hydraulic fluids, and additional drilling fluids will be routinely discarded. investigators can also anticipate that many of thesame - mit-erlhali-•ll'-.uged during the construction of the actual repository. • ... Th e,.Aq .•.Pepository is-completely- sealed, significant amuunts of organic materials will be located near it. iOur"speeific~concirnwis: that, these organic materials may be used6 as growth"' '-- substrates by large numbers of microorganisms, which in turn may influence the transport of radioactive elements from the repository. Microorganisms can affect transport in one or more of the following ways:
-1- ,
r 1) Alter the cuzvposlt'ioniof the groundwaterý chemistry through changes in pH orEh 2) solbilize radioactive -element s by•producing chelating agents, 31) týrt•i rt the radioniIfIde by b61-ogical movement, 4) transport the radionuclide by colloidal dispersion, or •5) retard the transport of the radionuclide by sorption onto a non-motile solid phase. I The microbiological research being performed as part of the NNWSI Project can be divided into three sequential research tasks. First, we must determine the susceptibility of drilling fluids and other exogenous materials to . microorganisms indigenous to Yucca Mountain. Second, we must ascertain the potential for sorption of radioactive elemenfs bi'these biodegrading organisms. Finally, we need to document the .effect- of microorganisms on transport. The results of the first and second tasks are presented in this paper, and research on the third task has recently been initiated. As stated earlier, during the exploration of Yucca Mountain as a possible location for the candidate high-level nuclear waste repository, several million gallons of drilling fluids were lost. Table I contains a list of the drilling fluids lost in wells-located within a 3-km radius of the G-4 well, I the proposed location of the Exploratory Shaft. TABLE I
LOCATIN ND VOLUES OF DRILLING FLUIDS LOST NEAR WELL G-4
Well Location Quantity -' Characteristics USW H-1 Drill Hole Wash (DHW) 582,710 gal Detergent/Water 1:133 USW H-3 Block 322,325:gal •DA 1:60 USW. H-4 Block 418,117 gal D/W 1:325 UswUSW 11-6H-5 Block 711,782 gal D/W 1:141 DfNWest of Block 931,811 gal D/W uSW G-1 2,600,000 gal Polymer USW G-4 Block not available Polymer UE-25a#l DHM not available Polymer UE-25a#4 DHM not available Polymer UE-25a#5 DHW not available Polymer UE-25a#6 DIM not available Polymer UE-25a#7 DHM not available Polymer UE-25bl H D2m 1,282,280 gal DW 1:300 UE-25wt#4 2 km N.E. of G-4 not available D/W uw wq DIOCK not available D/W The average number of gallons of drilling fluid lost was 978,432. This total does not include diesel fuels or lithium chloride fluids. Based on the data available for seven drill holes, we estimate that nearly 15 million gallons of fluids have been..intLrr&-d Into tthlg r~a6 178,432x 15...... 14,676,482). The drilling fluids lost were either-ASP-700 (Nalco Chemical Company), a polymer-based fluid.used-in-geologio4*ste holes.oI,6%22.Tr •., ._ Chemical Company), a detergent-based fluid used in the drilling of hydrology,. wells. The following is a list of the ingredients in each fluid:
-2- - 44-4ýý
iiw' * -.
. .
-1. Turco 5622 (Detergent) - 77-87% Water 10-20% Isopropyl alcohol <0.5% Gelatin <0.5% Ammonia <0.5% Sodium nitrite 2.0% Surfactant (linear dodecylbenzene sulfonic acid). 2. Nalco ASP-700 (Polymer) 43.2% Water 29.0% Polymer (acrylamide co-poly w/Na acrylate) 27.8% Light hydrocarbon a) Parafinic solvents (hexane, pentane) - 91% b) Emulsifier (or polyethylene glycol p-isooctylphenyl.ý ether) - 7.2% c) MTh (ethylenediaminetetraacetic acid) - 1.8% Both Turco 5622 and ASP-700 contain oxidizable energy sources. For the Turco 5622 isopropyl alcohol, gelatin and the surfactant are biodegradable. For the ASP-700, most of the light hydrocarbons can serve as energy sources. It is also important to emphasize that water from any of the Yucca Mountain wells contains salts necessary for microbiological growth. Table II was taken from the NNWSI report, "Groundwater Chemistry Along Flow faths Between a '-V. Proposed Repository Site and the Accessible Enr-.vir ment." In coumarison to the following list of constituents added to a minimal salts bacteriological medium, we see that there are ample salts in the groundwater at Yucca Mountain -to support microorganisms: f Salts EL-1 NaNO 37.5 75.0 Na' 37.5 20.0 'C CaC1 25.0 NaSib, .20 58.0 PH 7.0
*04M -MA ý : ::••;;• ••r
-3- I ' 1 1
'1 TABLE II .4 SA ANIO COCN1RATICNS IN GFATERS FROK TE VICINITYf O* YUCCA MNMIN Concentraaon (aqATt I i ii - • I I f mg Na "LL ja4 Ci i •j Usw VH-1 7.5• 1.5 80 -. 10 45 I Iusw H4:' 0.22 74 2100.12 ..0.04 *7.7 27.5 usw H1-3 7.1 J 1.1 0.01 124 1.5 0.13 0.01 8.3' 31.2 USW H-S:, 7.1 0.03 54 2.3 0.01" N.D. 5.7 14.6 0.15 56 2.5 0.04!i 0.02 5.5 15.7 USW Hw-4 7.5 1.6 ! rUSW H-4 7.4 K.10.8 <0.1 51 2.6 0.03I 5.8 19 7.7 0.19 84 0.005 6.2 23.9 UE-25blf 7.2 19.7 0.68 56 3.3 0.04• 0.004 7.1 20.6 UE-25b1. •18.4 0.68 46 2.5 0.69r 0.36 9.8 21.0 UE-25b#1 7.3 17.9 0.66 27 3.0 0.08f 0.07 6.6 20.3 J~13 6.9 11.5 1.76 45 5.3 0.041 0.001* 6.4 18.1 'UE-2912 7.0 "11.1 0.34 51 1.2 0.05 0.03 8.3 22.7 J-12 7.1 14 2.1 38 5.1 7.3 22 419 0 31.9 171 13.4 <0. i <0.1 337 129 6.7, !i .8 .1
11 ..4 4
'I 4 4i 4 p.• 4j . -4:.. 1 .4, • ::rL.
An inventory'of organia -materials that will be used during the construction .of the Exploratory .Shaft-at. Yucca Mountain is listed below. The inventory was prepared by V. Gongqof eynolds Electrical & Engineering Co., Inc. Diesel fuel Brake fluid AlT Engine, oilr Gear oil . Greases, including wire rope Rock drill oil/ tablebase Cleaning solvents Paints Antifreeze* Dust stupressat* Road ol (chip seal)* CaCls tire ballast-. Service water .tracersi sed for ýthe following processes: Drilling* - face, rockbolt, corehole, borehole -'Wetdown - muckpiles, dust control ....Drift-wall cleaning for geologists Structural backfill compaction Water-line leaks Scrubber cleanout water* Concrete (water,. sand, cement, aggregate grout, curing and form release chemicals, shotcrete, and additives) Human fluid waste Fire extinguishing -hemicals Explosives and explosive residue Drilling fluids -ES2 pilot hole.
*Fluids or chemicals that may have reasonable alternatives. Table III presents an:aproximation of the volume of fluid to be used during the Exploratory Shaft construction.
.- .. .7 - -
-
-5- * . ? 1 4
TABLE III t ESTMATE OF ELUID EXCPOSURmE r1JRx i EXPLOPAIME SHAFT CON~STRU~CTION Hydraulic Duration Oi Anti SRock Drill "(Weeks) Grease: Freeze Water Phase Surf U.G___ Description Surr'UlG. Surf U.G. Surf U •. 1. Surface Facility Construction Site Prepia ration 14 225 293 84 5,266,688 50 Facilities Construction 16 147 152 80 1,929,000 2 :,Shaft tinking ontractor A.. Collaring-& Headframe 255 295 "75 1,050,000 Shaft Sinking & Testing 31,32 517 740 431 158 4,410,000 89,040 . Station Con 220 . struction 26 452 109 611 465 130 1,820,000 187,620 Raisebore 312 ES-2 2'1 3 84 13 100 15 420,000 17,010, 3. Test Construction SSupport Excavation 52 884 884 1,357 1,62'8 260 7,280,000 1,084,29 Construction 12 154 84 288 ,20i8 60 420,000 Test Support 63,00 110 616 308 1,232 J85 8 220 3,850,000 77,00
-6 4,: i Most of the items on the.-nventory list are biodegradable and, therefore, as with the drilling fluid, will support microbial growth. We anticipate that large amounts of water will. be used for dust control. Although a list, has not yet been prepared, we can assume that many of the same organic materials will be used in the construction:of the candidate repository. The introduction of I.'these organic nutrients is an ongoing process, • concomitant to man's activities at the repository, and will not cease until the repository is sealed. the .ovement of organic contaminants in pu~surface environments has been the subject of several excellent reviews.' Generally, though, the factors governing the-transport of organic materials in soils are advection, dispersion,. sorption, retardation, and the partitioning of organic contaminants in the aqueous phase. We directed initial efforts towards confirming that those specific drilling fluids used at Yucca Mountain are susceptible to microbial attack. . Once- . -biodegradation waIs cfid'Z'5 research focused on identifying the bacteria isolated from the Nevada Test Site (NTS). Subsequently, we found these bacteria to biodegrade drilling fluids. Finally, studies were performed to determine the growth rates, of the microorganisms, changes in pH, oxygen use, and changes in the viscosity of the drilling fluids. We used this information to make concluding statements regarding the effect of microorganisms on ground water chemistry. II. BI S OGRDAIN•ISi w:
A. Sources of the ,_r.... There are several factorsthat influenre the extent and the duration of microbial activity at.Yudcp.Mountain. They are as follows: 1) Water. -Therea-iseo water (65% saturation) in the unsaturated zone to support microbial.ogr-wth. 2) Mineral Content.-' The mineral content of the waterýis capable of., supporting a microbiological population. 3) Organic Matter.- There is very little organic matter present, suggesting that little, if any, organic nutrients exist. Heterotrophic microorganisms probably exist in the unsaturated zone at a low level of existence (low numbers and metabolic rates). In all probability autotrophic microorganisms exist in the subsurface environment of Yucca Mountain, using reduced; inorganic compounds such as FeS., H2 and S as sources of energy. The limiting factor is the low nutrient, or energy source, level. with the addition of nutrients, microbial activity will increase. The duration of higher activity w1ll deppehd on the concentration of energy source transport of organics along the flow. path, the presence of inhibitors, flow rate, and the build-up of toxic by-products. Microbial activity is; s.imply a function of nutrients and will occur only when n=trients exist.. Be;.aiseof metabolic products, the effects of microbial activity will be a very long term factor. For example, humic acids are produceq by microorganisms,:• are recalcitrant, and are known to combine with metals. To isolate microorganisms capable of biodegrading drilling fluids, soil samples were collected from*two drilling locations at Yucca Mountain (USW H-3 and UE-25c#2). Because" these soils had received discharges of'dfillng-fl~ilde • during the course of the drilling operations, we felt these locations were appropriate choices. Soil samples were placed in sterile 500-ml, wide-mouth plastic bottles and were imediately returned to the laboratory for analysis. The following mineral salts medium was used to culture these microorganisms:
- 7- Stock g or ml/ Salt (a-/00 ml !Lo) 1.5 g 5 ml 1.5 5 ml 3.0 5 ml 1.5 5 ml 0.8 10 ml 0.5 Na, S0 3 H2 0 10 ml Trace elements: 1.16 H3 903 10 0g 50 pg 98 41 mg mg ZnCl2 5 Col mg 6H2 0 2 mg 4 g Agar 15 iI0pH - 7.2 1000 ml To 1.0 1 of this medium, either 5 drilling fluids ml of Turco 5622 or5 ml5of ASP-700: was added. Because the drilling fluids were the source, growth on this only energy. medium could be interpreted as biodegradation of these fluids. Solid media were inoculated with washings following of the soil samples and, two weeks of aerobic incubation at room temperature, :.bacterial colonies were growing several vigorously on the medium. To obtain pure cultures, isolated colonies were transferred to fresh an additional media and incubated for' two weeks. Colony morphology and microscopic characteristics were then determined, and this information is presented in Table IV.
ThBLE IV
CHARACTERRZATIO OF COLCY AND MICIOSCOPIC ORPHOLOGY Culture No.' Colony Morpholoy Microscopic Morphology .1 Tiny isolated colonies, G- long, thin rods. b mixed culture. Blue ends. 2, : Large white colonies. G- rod. Short, almost cocci. Lens-like appearance. Sometimes 3 pairs. Similar to No. 2. Could Same as No. 2. be contaminated with No. 1. 4 Small, isolated collonies, G- long, thin rods. similar to No. 1. Pure. No blue ends. Same as No. 3. Girola of varyinNo.eI. '' Similar to No. 1. No: growth. 7 Similar to No. 2, bxut not G- cocco-bacilli . .. as white. 8 with contaminants. Similar to No. 1, n iot G- rods, medium long and contaminated. thin. Large groups.
-8-
*r P' t- 5-
Culture No.2 .Colony MorphOloýx microscopic Mlorphology.,' - 9 Many isolated small colonies, G- small, thin, rods. no color. Contaminated with G+ cocci. 10 Odd growth. Colony in groove G- tiny, thin rods. of inoc. needle. ?lucoid. In large groups, randomly. White. 11 Mucoid growth, only G rods and- coccii ". fHeav initial inoculation. White. encapsulation. 12 Same as No. 11, but not as mixture of G- rods and Ge. much growth, cocci. Same as No. 12. G- fat rods. Encapsulated. 14 Heavy growth. Swarm-like, G- rods. 1) short, thin; spreading out from initial 2) shorter, fat (with bluie. inoc. ends). 15 Similar to No. 9. G- rods with G+ cocci/rods, 16 Heavy growth. ?lucoid white Similar to No. 15. colonies.
1 Nwbea, -8are cultures growing on Turco 56,2, 9-.16 on -. is "gram negative, Gei. is gram positive ASP 700.' .- 2Clearly, several species of microorganisms biodegradinig indigenous to the NTS are capable of drilling fluids. in subsequent studies, it had been determined.. .that,many of these species are the same or that some specieswrrelytor ..mre species. Since then we have shortened the 9 of which list to 13 species of bacteria, biodegrade ASP-700 and 4 that biodegrade Turco 5622 fluids. From these species, seven were sent to Dr. Myron -University Sasser's laboratory at the of Delaware, Newark, Delaware, for further identification, Briefly, the results P're as follows: Most Likely _mat Simni1 brj ~t
9C Pseudomonas stutzeri 0.461 Au b 0.154 9 0.253 Psi5To moms 0.056 0.047 !ýgobaterium tume faciens AlteromonasPuea~ciens 0.049 lid 0.281 As can b~e' seen, the similarity among the species that we sukzmitted and the reference- species is quite low -poorlain,. (inwhich 1.0 is perfect correlation and 0.0 is., In addition, we submitted organisms 11~ under. t66- different. i~civd different results. We tend to believeýthait't~irf'ciiscrepancyý" isue ~~-difficulty in-bacterial taxonomy associated Aith the genus. 'uooa, and also believe Dr. Sasser's staff. di jo.- Z.i~.dirbe, Aattempting, tidenitify these bacteria. Because imost of our studies. with the 11~ naveý de'alti. bacte riumu, we have done extensivetaxon c-aialyses ofth species. Table V expresses th.a results cf our inrvertigation.
-.9 - TABLE V
COMPARISON OF ORGANISMS 1l1 7T OTHER PSEDOMONAS SPECIES
p. P. P. P. aIcali- Tnendo- aero Tet -stutzeri genes cin ginosa li--rology (rods) 7,- ,--'--. motility (very motile) + + + + + Gram-stain - - GrowthO4C . ... Growth @ 43C + + 410C 419C 410C Poly-0-hydroxy butyrate ..... Egg yolk reaction ..... Fluorescent pigment ..... Starach hydLolysis + + - - Arginine dehyd-olase + (weak) - + + + Gelatin hydrolysis + - + - + Denitrification + - + : +
Carbon sources Dextrose + + - + Geraniol - - - + + -L-valine:- ...... - + Maltose + - - D-mannitol + d - - + Acetate + + + + + D-L malate + d d d d Ethylene glycol + + - + Butyrate + d + " + In addition to the above analyses, 11C was found to grow on the following substrates: propionate, stearate, maleiate, citrate, mannitol, ethanol, pyruvate, casein, casamino acids, glycine, aspartate, yeast extract, choline, betaine, Tween 80, Span 80, and Tween 61. Ethylene glycol (Table V), Tween 80, Tween 61 and Span 80 are components of the drilling fluids used at Yucca Mountaid. No growth was observed on pthatate, sorbitol, tartrate, EDTA, oxalate, sucrose, benzoate, nicotinate, cellulose, polyethylene glycol (3550), acrylamide, methanol, and pantothenate. Also, 11 is a strict aerobe; and in the complete absence of oxygen it does not fermenA glucose, nor does it grow with acetate as the energy source and nitrate, nitrite, iron (III), sulfate, or sulfur as electron acceptors. our examinations lead us to believe that microorganism ll-is -a strain of ,_- . Pseudomonas stutzeri. .The other microorganisms are closely rlated to those. species suggested y- Dr.. Sasser. The identification of those microqrganird-, found to biodegrade the drilling fluids was important because that information will aid in understanding the types of sorption being performed by the -wo.w-- microorganisms and in understanding the potential for metal mobilization.
B. Growth Studies It was not only important to determine which microorganisms are able to use drilling fluids as growth substrates, but also it was equally important to determine the rate at which microorganisms grow. Growth studies can provide useful information regarding the potential of the drilling fluids to support a
- 10 - .,population of microorganisms, and the duration of microbial activity.,•ý4 Two methods were used to determine growth rates: optical density and oxygen uptake measurements. Optical density measurements were performed on those species growing in Turco 5622 medium (the Nalco ASP-700 medium:is too.opaque to transmit light), and oxygen uptake measurements were performed on both drilling fluid media. For the optical density measurements, 5.0 ml of a 24-h culture was added to 25 ml of sterile medium in a 250-ml side arm-flask., Readings were taken every 24 h for 144 h (6 days) by using a Klett-Sumzersofl phottert."- Data are presented iq Klett Units (Fig. 1), for which 10 Klett Units represent approximately 1 x 10, bacteria per milliliter and 30 Klett Units: represent •' approximately 1 x 10 bacteria per milliliter. All optical density studies were performed at roo temperature (18-200C). In respiration studies, five drops of a 24-h culture were added to 2.5 ml of broth in a Warburg manometer. Oxygen use is determined by measuring the decrease in head space gas voluim over time. Figures 2 and 3 show the. results as micromoles of oxygen consumed per hour per milliliter of medium. All oxygen consumption studies were performed in a water bath at 206C. Both the optical density and oxygen use results demonstrated distinct growth rates. For the optical density measurement, the bacteria grew rapidly for 48 h and then growth slowly increased for the duration of the experiment. S..Oxygen consumption rates for the same species %how.-d a similar pattern. The greatest use of oxygen occurred during the first 6 h of growth. It should be noted that oxygen consumption does not correlate directly with optical density at .... because the greatest use of oxygen occurs when the optical density, is low, the early stages of growth where the young cultures are consuming 0 at a much higher rate than older cultures. Although there was a temperatur~e.aifference.. between the two experiments, it probably was not enough to make.a real.. difference in the results. 7he following data were obtained from earlier experiments done t cultures: . Hours lh * 24 893 48 663 72 603 96 451 120 404 144 222 168 75 . 192 15 • 4 With time, the rate of oxygen use decreased. During the entire exper S8.14 x 10 pM1of oxygen was consumed per gram of drilling fluid (per 192 h). -Because one gallon of drilling fluid (diluted 1/42 with water, the dilution the drillers at Yucca Mountain) contains 90 g of polymer, we anticipate used by7that'.31. M 0 would-b consumed forevery- gallon of plymer,-undery,optimal .. - 7.30.8conditios-(8.12.x-1O-i'-O./g.x-90-g/ga.73.8 x 10 pn, ;/br*,:oa7.31 M of gallons of driliing fluids have been 0 2/gal)., Considering that mfillicns used during the site characterization process, there is a strong potential for Woxyge•depletiorand- a- concomitant drop in the oxidation-reduction potential.-.
.C. Aviscosity long-term experiment was performed investigating viscosity changes of ASP-700 and Turco 5622 medium. Five bacterial species were grown individually - 11 - .. , V10
.• -"-0---.-- ... 1
0S 48-7
"0 ", 48 72 . 96. O 144 -. 16 Hours
Fig. 1. rovth.of three bacterial isolates In Turco 5622, measured by a Kle mers6o photometer.
S...... = 6 30 54 78 102 126 150 ri HOURSA ..
Fig. 2. Oxygen consumption of 'three bacterial .isolates..in ASP-70.06, measured by Warburg manometeri
741 10000 #51 isl NI#81 1000 ' I
= Ii .A. S-,10 't: 1 I I I
6 30 5+ 78 102 126 10 HOURS I
*Fig. .3 Oygen consumption of three bacterial Isolates, in Turco 5622 measured by Warburg uaaometers. -in the -AS~-700 medium, while two species were grown in the flirco..5622 medium.,--,, . For each species, 180 mf of medium was incuated with 18 ml of a 48-h culture. These flasks were mixed at 60 rpm for 25 days at 229C. T7wenty-five milliliters of media was withdrawn on days 0, 3, 7, 12, 18, and 25 for viscosity measure ments. To,.measure the viscosity of the ASP-700 medium, a small glass bead (2.9-an diameter, 0.030399 g) was dropped through a glass tube (11.4-mu inside diameter by 20 cm) containing the medium.' ,The .fall of the bead through the 20--ca distance was timed, and any change in traviel time was assumed to be -t indicative of a change in viscosity,.relative to nonk-inoculated control. For "-ihie'T&ic 5622 medium, the rise of air ýbubbles throuigh the medf'tzi was ia measure viscosity. Air bubbles were injected into the medium using a 10-pl, precision syringe and, as with the ASP-700 medium, the travel distance was 20 cm. The results of this experiment..are presented in Table VI. Any increase in t~ie values reported over time can be interpreted as a relative increase in the viscosity of the medium. Conversely, as the numbers decrease with time, we could assume that the viscosity of the medium was decreasing. TA.BLE VI
THE~ EF FES OF BACTERIAL G1FUl CNTH VISCOSITY OF ASP-700 AND.TURCO 5622 nzI12?
P ASP-700 Turco 5622 9 As 11AX I _ A A "Nil 9A`3 ___ 0 0.93 0.93 0.90. 9 .92 0.90 1.32 1.24 3 0.94 0.99 0.96 .0.94 0.95 1.01 0.98 7 0.91 0.98 0.92 0.91 .0.92* - 1.25 12 0.92 0.99_ 0.95. 0.90ý --0.90 1.33 1.19 18 0.7? 0.75 0.74 0.71 0.70 1.51 1.75 25 0.80 0.84 0.86 0.75 0.75 1.33 1.21 aResults are expressed as a ratio of the unknown value divided by the control value. Apparently, as the bacteria grew in the ASP-700 medium, the viscosity decreased. This was true for all five of the species that were tested. Following an initial decrease, the viscosity of the Turco 5622 medium tended to increase with time until day 25 for which the values were similar to day 0. These results suggest that more than one constituent of the Tutco 5622 medium was being acted upon by the microorganisms, which in turn had a varying effect on viscosity.
9S7.98 P. mendocina 7.72 9 A5 bC
- 15- Species pH Assigned~-Species
"A ~8.06 A1 8.03 P. stutzeri 9 h2b 8.30 P. mendocina. 9 N.CH.. 8.23 P. mendocina 1{~o 5%62 contro 76 stag pond "B* org 2. 7.73 6 7. 69 1A 7.59' A. ttintfaciens Stag pond #2 7.66 A.- putrefaclens
9 with few exceptions, bacterial growth had little effect on pH. Species .2b. and 9 both increased the pH of ASP-700 medium by 0.2 and 0.3 pH units, respectively. E. Survival. A siiple experiment was performed to determine the longevity of microorganism l11 . oe milliliter of a 24-h culture was added to 50 m.lof_. nutrient broth ;nd maintained at room temperature. Optical density and colonry-forming unit measurements were tecordeu periodically over several.. ,ult-rew-~s~inoz a~ted.,OD-october'lO0, 19850, and as.,_of-.., months. _The. initial that December 30, 1985, the study was still being conducted. We have estimated this microorganism will survive in nutrient broth for over 2 years. siga light microscope, .ýwe. observed that a~fter. 18 h of incubation,.: the :cell' mrphoblogy of this culturechaniged'~frogra rod to- saltalrod;or cocci This shrinking of bacteria was consistent with the finding of Humphrey et al. who have reported "dwa'rfism" in starving marine bacteria. The bacteria,.' stage as a means of coping with a harsh environment,. mnd -apparently enter-this of . there may b- a relationship between the decrease in size and the survri~ia3 the bacteria. is.'Wie*Af7l These findings indicate that this bacterium, a Pseudcmnas suited for long-term survival at the Nevada Test Site. Ill. SFTCZ4C STUDIES of 71 Microorganisms are knc , to accumualate a variety of metals as a means protection against the toxic effects of the metals. The simplest protection. mechanism used is the precipitation of the metal on the outer surface' ofth microorganism. This precipitation occurs because of the activities of membrane-associated sulfate reductases,' or through tF biosynthesis of oxidizing agents sucn as oxygen or hydrogen peroxide. In this way, metals such as iron are deposited on the surfaces of many species of bacteria, uranium on the surface. of fni and iron, nickel,. copper.,. alumninum,. cudnd ichr umon W,~ surface, or algae. X o the~ce tthe ptakey_15pthcteria and green algae. iive" 3 -ý,_,-be f awxbtoc'nce trate4Lckel 000times mre than the, original' metali of metals proves-ITYbd rather specifid dthifi the culture..med~iium. The binding ý,. c-harge, .ionic radius, and coor'dination geometry thie priidcminiimnt factors-. metals from reaching%"- -- Another measure -adopted by miicroorganisms to prevent toxic levels is the biosynthesis of intracellular traps for the reimoval of metal ions from solution; for example, the biosynthesis of the1julfhydryl containing metallothionein protein to bind cadmium and copper.
-16- By, depositing metals, -internally- or. externally, microorganisms are-not rily protecting themselves from the toxic effects of the metal ions, but are also, in effect, concentrating the metal in the biosphere. Because of the ztrong evidence in the literature, it is ouflcpcern that microorganisms are also capable of sorbing radioactive wastes. Strandberg et al. have described the intracellWar accumulation of uranium by the bacterium Pseudomonas aruginosa. Interestingly, the uptake of uranium is quite rapid, as if mediated -anactive transport -system even though a uranium transport system has not yet beenýdescribed& Intthexcase.of plutonium, Neilands and Raymond et al. have suggested that it could be moved int the cell via the well-described siderophore iron trasport mechanism.' The sidfrophore '.,ansport system could be moving Pu in an analogous manner to Fe , because cjL a similarity in their ionic radius/charge ratio. Because of the evidence presented above, we investigated the effects of -icroorganisms on the uptake of radioactive wastes, particularly from the standpoint of enhanced metal mobilization. A) media. Bacteria were grown and maintained on a minimal salts medium .that contained 1.0% of the ASP-700(w/iV) drilling fluid, which is used as an energy and carbon source. -3. B) Pu Feed Solution. Twenty-five milliliters of 0.1 m 2gPu4+ solution tracer developed at Los Alamos Natimoal Laboratory was transferred into a washed Pyrex centrifuge cone containing 1 ml of saturated NaNo ýulution. The test tube was then4 laced in a heater block until the liquid had evaporated completely. The Pu. '-residue was resuspended in an appropriate amount of 0.1 N HCI and transferred ti6-'• washed 50-ml Oak Ridge-type, polycarbonate centrifuge tM. t?.aiZ!dry.Filtered (0.05 im) J-l•-1ter well used to. resuspend, the P•.,Pu :ýt•:;•:! fifnal concentration of 10 to 10 M Pu. The oxidation state of the Puwwas pred inantly 4+, although same 5+ and 6+ may have also been present..`:.._, C) Sorption. The Pseudomonas pR was transferred from a streak plate (nutrient agar, Difco Co.) to a 25D-ai Belco cultuze flask containing 50 ml of nutrient broth (Difco). The flasks were incubated at rocm temperature for 18 h. One milliliter of broth wastrsferred to another 250-mi flask containing 49 ml of nutrient broth. Again, the culture was incubated at room temperature for 18 h then used to inoculate fifteen 250-ml Belco side-arm flasks, each containing 49 ml of nutrient broth. The flasks were incubated at room temperature for periods of 3, 6, 12, 24, or 48 h. After each incubation period, we removed three flasks and determined the optical density of each culture by using a Klett-Summerson photometer, which was equipped with a #54 filter (520- to 580-nm transmission). Wei then determined the number-of.viable cell counts (or colony-forming unitsg).ý sff itdard-dilution and pour-plate methods. The total broth volue from-each side-arm flask was transfcrred to a 50-ml polycarbonate Oak Ridge-type centrifuge tube and centrifuged at 17,300 g for 10 min. The supernatant was discarded and the bacterial pellet was washed with 10'ml of phosphate buffer. The tubes were again centrifuged at 17,300 g for 10 min. This process of washing:the pellet was performed a total of three -mm w fes-After'the final washing,-the supernatant was discarded and 20 if 6f' Pu feed solution was added to each of the three tubes. The tubes were agitated at 100 rpm at room temperature' -for 5,.days... The tubes., erecentrifuged...' at 17,300 g for 1 h, then tha supernatant was transferred to the Oak Ridge-type 50-ml centrifuge tube and centrifuged again at 17,300 g for 1 h. The pellets were air dried at room temperature and weighed. Following the 1-h centrifuga tion, 10 ml of supernatant was transferred to another Oak Ridge-type centrifuge tube and centrifuged at 17,300 g for 2 h. Five milliliters of supernatant was
- 17 - which 5 ml of water removed and transferred to a 20-ml scintillation vial to and 10 ml of Insta-Gel liquid scintillation cocktail (United Technologies, also added. The vials were mixed and placed in a Seale Packard company) were 1 to counter fw 50 min. The window opening was set at Mark II scintillation was set at F; and 99 minimum. the maximum gain was set at 000; the range switch the scaler,-,as. turno~~~ ~~iin vial de-Cermined'three D) .Si aotoU.d _The activity of each was ratio (activity in solid times. These values were averaged and the sorption phase) was determined as follows: Background count was phase/activity liquid liquid from the experimental count, yielding the activity in the subtracted feed subtracted the liquid phase activity count from the phase. we then We count, yielding the activity in the solid phase. solution activity of the initial the solid phase activity count by 4 (because only 5 multiplied of the dry of feed solution was counted) and divided by the product 20 ml phase. The written weight of the bacteria times the activity in the liquid equation reads as follows:
4 x (activity in the solid phase) Ratio - (activity in the liquid phas Sorption S(dry weight) x (activity in the liquid phas-)
three replicates for each experiment, with each experiment We performed ratios, times. Although our data are presented as sorption repeated three is actually there exists no information to demonstrate that sorption observed is the bacteria removing the plutonium from occurring. What we are able fese experiments serve to demonstrate that these bact-ria solution. 2 regarding the mechanism to remove Pu- from solution, and are not-definitive rate of removal.. or optical Bacterial Growth. Table VII presents the data for the E) the bacteria. Each set of densities, dry weights, and colony-forming units of that the Pseudomonas sp. grew rapidly, reaching measurements demonstrates numbers .im1-1 (or cells/tl) in 1i h of incubation. The cell 2. x 1 cells' this species was slowly increased to 3.4 x 10 cells ml in 48 h. Clearly, concentration over a prolonged period of time. able to maintain a high cell we these cells were examined under a light microscope, Interestingly, when 12 h, the cells over time a change in their morphology. At 3, 6, and observed the cells decreased in were rod shaped; however, after 18 h of incubation, became cocc?19 in shape. size and VII expresses the Sorption of Pu' by the Pseudamoa sp. Table F) most outstanding feature of results of the sorption experiments. Pyfha~pthe Pu by Pseudomonas sp. The bacteria.. data is 'the.stg~ng4 sorption of the tihe sterile control concentratedthe the Pu.. nearly 1000 times greater tFhn also appears to be sterile, crushed tuff, 75-200 mesh). +•Sorption .. .. related(1 g of to the Orysiological .. state of thebacteria. On a per-gram, dry-weight closely by the 6-h basf e.T-t&M ltuieý sorbed the most Pu , followed the lowest sorption ratio for the 12-h and 24-h cultures.•-,,We0b6VrM by the 48-h cultures. cultures, fo.1.oedi, by.a slight increase in sorption
- 18 - TABLE VII I OPTICAL DENISITIr DRY WEIGHTS, COLCW4-FORMNG UNITS AN I SORPTICI RATIO, FOR A PSEUWCMNAS SP. rVRING 48 h or InCU)BTICtN
ati D tX Sorption I 1W Unts) (per ml)(m/±ad
3 7 7.53 0.00080 l.Ox10 9,280 + 707 6 26.8 0.00179 8.8x10' 9,840 ±4,417 12 1174 0.01411 2.lxO', 2,156 161-, 24 .173 0.01474 227x10' 2,080 ±230 48 155 0.01547 3.4xlu' 4,300 ± 342 Control.,- -~ 1.000 - 0.56 (1 g crushed tuff)
IV. DISCIZSOc OFý BICDE~tVCNq AM SCI~N The, majorz..results of our; biodegradation investigations show that the millions of'. gallons of drilling fluids used 'at Yucca biodegradablep, PMoutain are 1 that. these drilling fluids will support a large population of,;.: microorganisai' that use measureable amounts of oxygen; are capable and that these bacteria of surviving for long periods of time. The presence of microorganisms can have profound effects on the quality of groundwater chemistry. Both inorganic and organic chemicals environment entering the groundwater can be ýtransformed by microbiological processes. To obtain energy for growth, microorganisms can oxidize organic ccuxpounds, hydrogen or reduced forms of iron, nitrogen, or. sulfur. Electrons from these oxidation reactions are transferrid' to electron acceptors, such as oxygen under and nitrate, aerobic conditions sulfate,' and carbon dioxide under anaerobic conditions. In addition to transforming chemical crnpounds, microbial growth can change the by-ch&educton cages can1 potntialof the system.. Such.. q, oi~ti j chZemical phenomnena, such as precipitation or .. :-.dissolution of- phosphatesl--and heavy metals or chemical of iron oxidation or reduction and sulfur 'salts. Thus, many physical axi,, rach pian, beth ýr~brought abdui 6idotiaE 'wth-the- deccxzý6stion or" transforimitioso organic and ~pr nc i_bymaterials microorganisms living in- th~ub e 7rn~i The intent+of 'ourrserc was not to invest-igate microbiological the direct effects of growth on every aspect of groundwater chemistry; such an investigation was beyond the capabilities of the NNKSI of microbial Project. The effects activity on groundwater chemistry are well documented in the
- 19 - *.
literaturi of Britton and Rheinheimer.29- 3 Rather, our investigation demonstrated-ther.a.is.potential for significant and long-term microbial activity in the groundwater at Yucca Mountain. The investigation also emphasized that strong scientific evidence indicates that such activity often has a profound effect on groundwater chemistry. Based on the information gained from the literature and our laboratory studies, the following ,-7Proximations can be made regarding water chemistry: 1) .z There-exists no -evidence-that-microbial activity will change the PH of the groundwater. All of our studies regarding pH resulted in little or !c change in the pH of the growth media. In addition, the concentration of bicarbonate in the groundwater serves as a strong buffering agent. Therefore, nu- should be unaffected by microbial activity. 2) Oxidation-Reduction Potential. There is a strong possibility that T-crobiological activity could result in reducing conditions, at least in lo calized environments. Our experiments have consistently yielded information confirming oxygen uptake. However, we have little information regarding the ,ng-term stability of reducing conditions. 3) Viscosity. Depending upon the drilling fluid, viscosity is either increased-or-decreased. Because viscosity, as affected by microbial activity, is also dependent upon the species of bacteria, it would be very difficult to "•... : M.V:It'tain.:redict the overall effect of microbial activity cn viscosity at Yucca Thus, the most important effect that micrc~rganisms may have on the groundwater chemistry of Yucca Mountain is the removal of oxygen and the concomitant chemistry in oxidation reduction potential. During our sorption studies, it is interesting that we never observed the classical log death phase of organism growt.h. Normally, researchers would anticipate that after-24-h, because of nutrient depletion and the build-up of toxic by-products. cell.viability would begin to rapidly decrease. The bacterii?,! however, increased- in numbers throughout the duration of the "experiment. The observed shrinking of the bacteria is consistent with the finding pf Humphrey et al., who have reported "dwarfism" in starving marine bacteria. The bacteria apparertly enter this stage as a means of coping with a harsh environment, and there may be a relationship between the decrease in size and the survival of the bacteria. The possibility that the bacteria may sorb the 39 Pu4+ is not surprising because, as previously mentioned, bacteria are known to sorb many different metals from solution. These results are consistent with the findings of Strandberg et al. who rported a strong intercellular uptake of uranium by Pseudomonas aeruqinosa, The most interesting finding of our study is that sorption appears to be dependent upon the physiologigfj state of the bacteria. The results show that the younger cultures sorb the Pu more strongly than the older cultures. At 3 h, these cultures were entering log growth phase, - during-which cells rapidly grow and1 divide.-. At. 6 h,- the cultures entered log- grow`% herehase.and' again, the . Pu sorption was quite .high; however, by .... h P. 'rptiono12 declined rapidly. These events appear to coincide _with - -the oet o to ase` during which grt a d s no lccur and'deathl6l:°f the` cells has not yet begun. At 24 h, the sorption rate was-also l... andy-as-indicated bythe: cell number and optical density measurements,; the- . S...... cultures;were.in stationary. phase. At 48-h. t culture-were•-. • " ,..i stationary phase but for some poorly understood reason sorption began to increase. This observation is supported by the results of preliminary experiments, during which sorption was quite strong at 54 h of incubation. One
- 20 - I
-" ~~'Ai . . •.. . . :•ý possible -xplanation could be that after prolonged growth, the organisms have entered severe nutrient depletion and are2 jvor.ing much of their energy to mineral uptake, resi'lting in the j.lb°ingbe 'kenup' in a manne r anal oU.. ", .,:cation.The significant .prption coefficients obtained-ý -ý..--... to the essential Fe* 2 taken up similarly'.. by the 3- and 6-h cultures may also be oduo. Pu being I to a needed mineral. Alternatively, the . Pu may be toxic to the cells and the cells are more capable of"internally sequestering the plutonium at 3 and 6 r -h thanwthey are at 12 and24 h. Finally,.the sorption could also be the result -of a surface precipitation'mediated by. an .electromagnetic effect or accuwmla tion-by. a.protein. Witli:h ag the surfF characteristics, of a bacteria change, I and this change could. strcigl~y -affectý:,;P sorption. Regardless of the meansiby wchk* Pu is sorbed by the bacteria, the fact remains that these bacteria are able to remove this actinide from solution. What remains to be determined is the overall effect that this biological sorption may have on the movement of radioactive wastes from a high-level nuclear waste repository. Studies show that bacteria will be removed from suspension by soil or rock. In most classical saturated flow conditions, this is probably true; however, it is unclear what effect the surrounding rock will have on'.the bacteria if saturated or unsaturated fracture flow conditions are predcmina.t.Most-bacteria are motile and, therefore, are not conffned to an isolated location.' .We must emphasize that there exists little informationpregardift- the• extent -.of miýrobial activity in the deep, subsurface environmnt (B."dibirse, CorneillUniversity, personal ccmmzmication).- It has' always •been assumed that microbial activity occurred only in the first few meters below the soil's surface. However, recent advances-in sampling te-hachniques' hv~e kindled new interest in the subject, and iti ~ known mirba.atvt can, occur .to. depths. of .30. m; -or mre below the su'rfai UWfotonately,: .these sampling techniques cannot microbial acti4it eat'-a"er depths,"therefore, the extent of microbial ::"".access " activityat the" depths°otk'andate high-level nuclear waste repository is "still subject to considerable' cbnjecture. -. . . What we nave learned so far'-in our studies of microbial effects on radioactive wastes is considerable. We know that 'several species of bacteria are capable of aerobic-biodegradation of the drilling fluids used at the Nevada Test Site, andwe. knowthat at least one of the species is capable of strongly sorbing Pu 4 . What researchers need to investigate is the fate and mobility of the sorbing bacteria' in the envirorment and the extent of radioactive waste sorption.among other members of the microbiological community. once these two areas are more clearly understood, we can begin to make meaningful predicti srgding-the contribution of microorganisms to the retardation of radioactive7eastes in tuff."
- 21 - 1. Montazer, P. and W. E. Wilson, "Conceptual Hydrologic Model of Flow in the Unsaturated Zone, Yucca Mountain, Nevada," U.S. Geological Survey Water Resources Investigations report 84-4345 (1984). 2. Ogard, A. E., J. F. Kerrisk, "Groundwater Chemistry Along Flow Paths Between a-Proposed Repository Site and the Accessible Environment," Los Alamos .ationalLaboratory Report, LA-10188-MS (1984), pp. 9-10. 3. AndersonaM.M,.. Crit. Rev. Environ. Control 9(2) (1979), pp. 97-156. 4. Bear, 3.*,Hdraulics of Groundwater (McGraw-Hill, New York, 1979), p. 139. 5. Freeze, R. A. and J. A. Cherry, Groundwater (Prentice-Hall, Englewood Cliffs, New Jersey, 1979), pp. 385-62.T 6. Roberts, R. V., M. Reinhard, G. D. Hopkins, and R. S. Summaers, "Advection-Dispersion-Sorption Models for Simulating the Transport of Organic Contaminants," in Groundwater Quality, C. Ward, W. Giger, and P. McCarty, Eds. (Wiley-Interscience, New , 1985), pp. 425-445. 7.- S1044-1050.Fitch-'A. and F. J. Stevenson, Soil Sci. Soc. Am. J. 48 (1984), pp. 8. Humphrey, B., S. Kjelleberg, and K. C. Marshall, Appl. Environ. Microbiol. _.45 (1983), p. 43. 9. Seigal,. B. Z., Science 219 (1983), p. 25b.. 10. Wood, 3. M., Chem. Scr. 21 (1983), p. 155. 11. Wood, J. M. and H. Wang, Environ. Sci. Technol. 17 (1983), pp. 582A-590A. 12. Strandberg, G. W., "Biosorption of Uranium, Radium, and Cesium," in I c of Applied Genetics in Polution Control, C. Kulpa, R. Irvine, and S...... Sojkav-Eds.-(uaiversity of Notre Dame, 1982), ppt•126-142-. 13. West, J. M., I. G. McKinley, and N. A. Chapman, "Microbes in Deep Geological Systems and Their Possible Influence on Radioactive Waste Disposal," in Radioactive Waste-Management Hardwood and the Nuclear Fuel Cycle, Academic, Vol. 3(1) (1982), pp. 1-5. 14. West, J. M., I. G. McKinley, and N. A. Chapman, "The Effect of Microbial Activity on the Containment of Radioactive Waste in a Deep Geological 'Repository," Proceedings of the Fifth International Symposium on the ... Scientific Basis for Radioactive Waste Management,-Berlin, -June, 1980. 15.,,Champ, D. R., W. F. Merritt, and J. L. Young, "Potential for the Rapid Transport of Plutonium in Groundwater as Demonstrated by Core Column Studies," Proceedings of the Fifth International Symposium on the Scientific Basis for Radioactive Waste Management, Berlin, June, 1980. 16. Treen-Sears, M. E., B. Volesky, and R. J. Neuf.ld, Biotech. and Bioengr. . 27(1984), pp. 1323-1329. 17._Christofi,-N., 3. M. West, andJ3, C,-Philp, "The Geomicrobiology of European Mines Rulevant to Radioactive Waste Disposal," British Geological Survey report FLPU85-l (1985). 1R. Pice, T. R. and V. M. Willis, Linnol. and Oceanog. 4 (1959), pp. 245-277. 19. Honda, Y., Y. Kimira, H. Kitada, and H. FukumitsuT-"Uptake-and . _,AgccmulatioIýofnRadionuclides.in-Seawater by Plankton," in...nual.Reporiný-Annual Report-!'ý"- Just. Atomic Power, Kinki Univ. 9 (1974), pp. 19-24. 20.Strandberg, G. W., S. E. Stumate, and J. R. Parrott. Appl. and Environ. '•'. .. . . • ; iik ~ b l o rl•l 1) '-( 1 9 8 1)•~'2 7 4 5 . -- "' • • ..,ý.1-Drgeher---_T . ,ý"v f-ljrxniiand Molybdenum from Uranium Mine Wastewaters by Algae," Masters Thesis, New Mexico Institute of Mining and Technology, Socorro, New Mexico (1981)..---
- 22 - S-4f
22. Sakagu.hi, T., T. Borikoshi, and A. Nakajima. 3..F.rment... Tec.ol. 56(6) - (1975), pp. 561-565. 23. Horikoshi, T., A. Nakajima, and A. Sakaguchi, Agric. Biol. Chem. 43(3) (1978), pp. 617-623. 24. Aoyama, I., x. incm,.and .YeIncue,3. Radiat. Res. 17 (1976), pp. 69-81. 25. Marchyulene, E. D. P., Ekologiya. 2-(1978), pp. 80-81. `26'. eilands, j. B.';' Microbial"'iron Meitabolism (Aca.demic Pre ,ss,,New York, 1974) p. 547. 27. Raymond, K. N., G. Muller, and B. F. Matzanke, "Complexation of Iron Siderophores: by A Review of Their Solution and Structural Chemistry and Biological Function," Current Chem. 123 (1984), pp. 49-100. 28. McCarty, P. L., B. E. Rittmann, and E. J. Bouwer, "Microbiological Processes Affecting Chemical Transformations in Groundwater," in Groundwater Pollution Microbiolom, G. Britton and C. Gerba, Eds. (Wiley-Interscience, New York, 1984), pp. 89-116. 29. Britton, G. and C. P. Gerba, Groundwater Pollution Microbioloqv (Wiley-Interscience, New York, 1984), pp. 65-88. 30. Rheinheimer, G., Aquatic Microbiology.(Wiley-mnterscience, New York, 1980), pp. 135-17U.
- 23 - BICADOGATICN OF DRILINGE FLUIDS: EFECT"S ON AVINIDE SON
Larry E. Bersman, LS-3
Abstract
A Pseudomonas sp., isolated from the Nevada Test Site, was used to study ) the removal of 239pu from solution. Bacteria were grown in nutrient broth, harvested and mixed with 10-8 M 2 3 9 Pu feed solution. The results clearly demonstrate that bacteria sorb the actinide. In addition, sorption appears to be dependent upon the .pysiological state of the bacteria, with the younger cultures (3 and 6 h) sorbing at a higher rate than the older cultures.
Introduction
During the process of characterization, by the Nevada Nuclear Waste Storage Investigations (NNWSI) Project, of the geology and hydrology of the site for a candidate high-level nuclear waste repository at Yucca Mountain, Nevada, several million gallons of drilling fluids were used. Within a 3-km radius of the exploratory shaft it is estimated that nearly 15,000,000 gallons of drilling fluids were released into the block (candidate site), or in the immediate area. 1 The drilling logs from these wells indicate that a significant amount of the fluid was lost in the Topopah Spring Member, which would be the geologic location of the repository. The Topopah Spring Member 1
) has been characterized as a welded tuff with a low permeability and a high content of fractures. Montazer and Wilson2 have therefore suggested that in this member, lateral movement may exceed downward rates, a statement that was substantiated by the recent discovery of drilling fluids in the USW UZ-i hole (air drilled without fluids). Interestingly UiZ-1 is located several hundred meters upgradient from the closest fluid drilled holes (US; G-1 and H-I) and its drilling was limited to the unsaturated zone, at a depth approximately equal to that of the repository. In addition to those drilling fluids, several other forms of organic
materials will be introduced into the block. During the construction of the Exploratory Shaft, items such as diesel fuels and exhausts, hydraulic fluids,
and additional drilling fluids will be discarded routinely. It can also be anticipated that uuch of the same materials will be used during the construction of the repository itself. Therefore, before the repository is completely sealed, there will be a significant amount of organic material located near it. Our specific concern is that these organic materials may be utilized as growth substrates by a large number of microorganisms, which in turn may influence the transport of radioactive elements away from the repository. Microorganisms can affect transport in one or more of the following ways: 1) alter the croosition of the groundwater chemistry through changes in pH or Eh, 2) solubilize radioactive elements by producing chelating agents, 3) transport the radionuclide via biological movement, 4) transport the radionuclide via colloidal dispersion,
5) retard the transport of the radionuclide by sorption onto a nonmotile
solid phase. 2 The microbiological research being performed as part of the NNWSI Project can be broken down into three sequential research tasks. First, it is important to determine if the drilling fluids and other exogenous organic materials are biodegradable. Second, the potential for sorption of radioactive elements by these biodegrading organisms must be determined. Finally, the effect of microorganisms on transport should be documented. The results of the first task have, for the most part, been completed and are present in the milestone report titled "Biodegradation of Drilling Fluids: Effects on Groundwater Chemistry". The second task is now being performed, and the results obtained to date are presented in this paper. Research on the third task is scheduled to begin in the near future.
Microorganisms have been shown to accumulate a variety of metals as a means of protection against the toxic effects of the metals. The simplest mechanism used is the precipitation of the metal on the outer surface of the microorganism. The precipitation of the metal occurs because of the activi ties of membrane-associated sulfate reductases, or through the biosynthesis of oxidizing agents such as oxygen or hydrogen peroxide.4 In this way, metals such as iron are deposited on the surfaces of many species of bacteria: uraniun on the surface of fungi; and iron, nickel, copper, aluminum, and chromium on the surface of algae. A second means of surface accumulation is through the production of extracellular ligands that comlex metals outside the cell and prevent their uptake. Both cyanobacteria and green algae have been found to concentrate nickel to 3000 times the concentration in the culture medium. The binding of metals has been shown to be rather specific, with charge, ionic radius, and coordination geometry being the predominant factors. Another measure adopted by microorganisms to prevent metals from 3 reaching toxic levels is the biosynthesis of intracellular traps for the removal of metal ions from solution. One example is the biosynthesis of the 5 sulfhydryl-containing metallothionein protein to bind cadmium and copper.
By depositing metals, internally or externally, microorganisms are not only protecting themselves from the toxic effects of the metal ions, but are also, in effect, concentrating the metal in the biosphere. Because of the strong evidence in the literature, it is our concern that microorganisms are 14 also capable of sorbing radioactive wastes as well6-1 9 . Strandberg et al. have described the intracellular accumulation of uranium by the bacterium Pseudomona aruginosa. Interestingly, the uptake of uranium is quite rapid, as if mediated by an active transport system even though a uranium transport system has not yet been described. In the case of plutonium, Neilands 20 and Raymond et al. 21 have suggested that it could be moved into the cell via the well described siderophore iron transport mechanism. The siderophore transport system could be moving Pu+4 in an analogous manner to Fe 3+, due to a similarity in their ionic radius/charge ratio.
Because of the evidence presented above, we felt it necessary to investigate the effects of microorganisms on the uptake of radioactive wastes, particularly from the standpoint of enhanced metal mobilization.
MRTEI•ALS AND MMEKEG
Bacteria. A bacterium isolated from the Nevada Test Site was found to biodegrade ASP-700 drilling fluid (Nalco Chemical Coapany). Based on the results of a standard API 20E test strip, the bacterium has been tentatively 4 identified as Pseudomonas fluorescens. A culture has been sent to the American Type Culture Collection for a more extensive identification. The bacterium has been maintained on ASP-700 medium (described below) at room temperature with transfers performed on a bi-monthly basis. Streak plate analyses were performed periodically to insure that the culture was free from contaminants.
MlNdia. Bacteria were grown and maintained on a minimal salts medium that contained 1.0% of the ASP-700 (w/v) drilling fluid as an energy and carbon source. The constituents of the drilling fluid were as follows (on a percentage, w/v basis): water, 43.2; polymer (acrylamide-sodium acrylate copolymer), 28.9; light hydrocarbons (hexane, pentane, etc.), 25.8; non-ionic emulsifier (50% TWen 61, 50% Span 80), 2.0; and ETA (ethylenediaminetetra acetic acid), 0.0135. The constituents of the minimal salts solution are as follows (milligrams per liter of distilled, de-ionized water): NaNO3 , 22.5;
KH2 PO4 , 0.15; K2 HPO4 , 7.5; MgSO4 .7H2 0, 7.5; Na 2 CO3, 4.0; CaCl 2, 5.0;
Na2 SiO3 .H20, 11.6; FeCl 3 .6H2 0, trace; -nCl 2*-4H20, trace; ZnCl 2 , trace;
COC1 2 *6H2 0, trace; Na2NoO 4 , trace; H3PO3, trace; and CuSO4, trace.
239pu+4 Fee Solutio. Tenty-five ml of 0.1 m 239Pu solution (Los Alamos
National Laboratory) tracer were transferred into a washed Pyrex centrifuge cone containing 1 ml of saturated NaNO 3 solution. The test tube was then placed in a heater block until the liquid had completely evaporated. The 239Pu residue was resuspended in an appropriate amount of 0.1 N HCI and transferred to a washed polycarbonate Oak Ridge type 50-.l centrifuge tube to air dry. Filtered (0.05 pm) J-13 well water was used to resuspend the 239Pu,
5 to a final concentration of 108 to 10- 239pu The oxidation state of the 2 39pu was predominantly +4, although some +5 and +6 may have also been present.
Sorptin. The Pseudomonas sp. was transferred from a streak plate (nutrient agar, Difco Co.) to a 250-mi Belco culture flask containing 50 ml of nutrient broth (Difco). The flasks were incubated at room temperature for 18 h while shaking at 100 rpm. One ml of broth was transferred to another 250-mi flask containing 49 ml of nutrient broth. Again the culture was incubated at room temperature for 18 h. This 18-h culture was then used to inoculate fifteen, 250-mi Belco side-arm flasks each containing 49 ml of nutrient broth. The flasks were incubated at room temperature for periods of 3, 6, 12, 24, or 48 h. After each incubation period, three flasks were removed and the optical density of each culture was determined using a Klett-Summerson Photometer equipped with a #54 filter (520-580 nm transmission). The number of viable cell counts (or colony forming units) was then determined using standard dilution and pour-plate methods (Standard Methods for the Examination of Water and Waste Water). The total broth volume from each side-arm flask was transferred to a 50-ml Oak-Ridge type, polycarbonate centrifuge tube and centrifuged at 17,300 x g for 10 min. The supernatant was discarded and the bacterial pellet was washed with 10 ml of phosphate buffer. The tubes were again centrifuged at 17,300 x g for 10 min. This process of washing the pellet was performed a total of three times. After the final washing, the supernatant was discarded and to each of the three tubes 20 ml of 239pu+4 feed solution was added. The tubes were agitated at 100 rpm at room temperature for 5 d. The tubes were centrifuged at 17,300 x g for 1 h, the supernatant was transferred to a 50-ml Oak Ridge type centrifuge tube and centrifuged again at 17,300 x g for 1 h. The pellets were allowed to dry in the air at 6 room temperature and weighed. Following the 1-h centrifugation, 10 ml of supernatant was transferred to another Oak Ridge type centrifuge tube and centrifuged at 17,300 x g for 2 h. Five ml of supernatant was removed and tranferred to a 20-ml scintillation vial to which 5 ml of water and 10 ml of Insta-Gel liquid scintillation cocktail (United Technologies, Packard Co.) were also added. The vials were mixed and placed in a Seale Mark II scintillation counter set at 50-min count time, window opening set at 1 to 99, maximum gain set at 000, the range switch set at F, and the scaler turned to the off position.
Sorption Ratio. The activity of each vial was determined three times. These values were averaged and the sorption ratio (activity in solid phase/activity liquid phase) was determined as follows: Background count was subtracted from the experimental count, yielding the activity in the liquid phase. The liquid phase activity was then subtracted from the activity of the feed solution, yielding the activity in the solid phase. The solid phase activity was multiplied by 4 (because only 5 of the initial 20 ml of feed solution was counted) and divided by the product of the dry weight of the bacteria times the activity in the liquid phase; i.e.,
Sorption Ratio - 4 x (activity in the solid phase) (dry weight) x (activity in the liquid phase)
Three replicates were performed for each experiment and each experiment was repeated three times. It should be mentioned that although our data are presented as a sorption ratio, there exists no information to demonstrate that sorption is actually occurring. What is being observed is the removal of plutonium from solution by the bacteria.
7 3EELTS
Bacterial Growth. Presented in Table I are the data for the optical densities, dry weights, and colony forming units of the bacteria. Each set of measurements demonstrates that the Pseudomonas sp. grew rapidly, reaching 2.1 x 109 cells ml-1 (or cells/al) in 12 h of incubation. The cell numbers slowly increased to 3.4 x 109 cells ml-1 in 48 h. Clearly this species was able to maintain a high cell concentration over a prolonged time period. Interestingly, when these cells were examined under a light microscope, a change in their morphology over time was observed. At 3, 6, and 12 h, the cells were rod shaped; however, after 18 h of incubation, the cells shrank in size and became coccoid in shape.
Sorption of 239pu4 by the Pseudmas. Presented in Table I are the results of the sorption experiments. Perhaps the most outstanding feature of the data is the strong sorption of the 239pu4 by Pseudomonas. The bacteria concentrated the 239pu+4 nearly 1000 times greater than did the sterile control (1 g of sterile, crushed tuff, 75-200 mesh). Sorption also appears to be related to the physiological state of the bacteria. On a per-gram, dry-weight basis, the 3-h culture sorbed the most 239pu+4, followed closely by the 6-h cultures. The lowst sorption ratio was observed for the 12-h and
8 TABLE I. Optical density, dry weights, colony forming units (CFU) and sorption ratio for a Pseudomonas sp. over 48 h of incubation. Optical Incubation Density Dry Sorption Time (Klett Weight CFU Ratio (h) Units) (g) (per ml) (mu/g)
7 3 7.53 0.00080 1. 0x10 9,280 (707 sd) 7 6 26.8 0.00179 8.8x10 9,840 (4,417 sd) 9 12 174 0.01411 2.lx10 2,156 9 (161 sd) 24 173 0.01474 2.7x10 2,080 (230 sd) 9 48 155 0.01547 3.4x10 4,800 (342 sd)
Control 1.000 5.37 (1 g crushed tuff) (0.56 sd)
24-h cultures, followed by a slight increase in sorption by the 48 h cultures.
DISC•SION
It is interesting that the classical log death phase of organism growth was never observed. Normally, one would anticipate that after 24 h, cell viability would begin to rapidly decrease, due to nutrient depletion and the build-up of toxic by-products. Rather, the bacteria increased in nmubers throughout the duration of the experiment.
The observed shrinking of the bacteria is consistent with the finding of Hmthrey et al. ,22 who have reported "dwarfism" in starving marine bacteria. The bacteria apparently enter this stage as a means of coping with a harsh 9 environment, and there may be a relationship between the decrease in size and the survival of the bacteria.
The possibility that the bacteria may sorb the 239 pu+4 is not surprising because, as previously mentioned, bacteria are known to sorb many different metals from solution. These results are consistent with the findings of Strandberg et al.,14 who reported a strong intercellular uptake of uranium by Pseudomonas aruginosa. The most interesting finding of our study is that sorption appears to be dependent upon the physiolgical state of the bacteria. The results show that the younger cultures sorb the 2 39 Pu+4 more strongly than do the older cultures. At 3 h, these cultures were entering log growth phase, where cells are rapidly growing and dividing. At 6 h, the cultures have entered log growth phase and here again the 2 39pu+4 sorption was quite high; however, by 12 h, 239pu+4 sorption rapidly declined. These events appear to coincide with the onset of stationary phase, where growth and division no longer occur and death of the cells has not yet begun. At 24 h the sorption rate was also low, and as indicated by the cell number and optical density measurements the cultures were in stationary phase. At 48 h, the cultures were still in stationary phase but for some poorly understood reason sorption began to increase. This observation is supported by the results of earlier, preliminary experiments where sorption was quite strong at 54 h of incubation. one possible explanation could be that after prolonged growth the organisms have entered severe nutrient depletion and are devoting much of their energy to mineral uptake, resulting in the 239Ru+4 being taken up in a manner analogous to the essential Fe 3+ cation. The significant sorption coefficients obtained by the 3- and 6-h cultures may also be due to 239pu+4 being taken up similar to a needed mineral. Alternatively, the 239pu+4 may be toxic to the 10 cells and the cells are more capable of internally sequestering the plutonium at 3 and 6 h than they are at 12 and 24 h. Finally the sorption could also be the result of a surface precipitation mediated by an electro-magnetic effect or accumulation by a protein. With age, the surface characteristics of a bacteria change, and this change could strongly affect 239pu+4 sorption.
Regardless of the means by which 239pu+4 is sorbed by the bacteria, the fact remains that these bacteria are able to remove this actinide from solution. What remains to be determined is the overall effect that this biological sorption may have on the movement of radioactive wastes away from a high level nuclear repository. It has been generally considered to be true that bacteria will be removed from suspension by soil or rock. In most classical saturated flow conditions, this is probably true; however, it is unclear what the effect the surrounding rock will have on the bacteria if saturated, fractur flow or unsaturated flow conditions are predominant. Secondly, it should always be kept in mind that most bacteria are motile, and therefore are not confined to an isolated location. Finally it must be emphasized that there exists little information regarding the extent of microbial activity in the deep, subsurface environment (Ghiorse, personal comunication). It has always been assumed that microbial activity occurred only in the first few meters below the soil's surface. However, recent advances in sampling techniques have kindled new interest in the subject, and it is now known that significant microbial activity can occur to depths of 30 m or more below the surface. Unfortunately, these sampling techniques are not able to access microbial activity at greater depths; therefore, the extent of microbial activity at the depths of the candidate high level nuclear waste repository is still subject to considerable conjecture.
11 What we have learned so far in our studies of microbial effects on radioactive wastes is considerable. We know that several species of bacteria are capable of aerobic biodegradation of the drilling fluids used at the Nevada Test Site, and we know that at least one of the species is capable of strongly sorbing 2P9u+4. What needs to be investigated is the fate and mobility of the sorbing bacteria in the environment and the extent of radioactive waste sorption among other members of the microbiological c~aznity. Once these two areas are more clearly understood, then we can begin to make meaningful predictions regarding the contribution of microorganisms to the retardation of radioactive wastes in tuff.
12 LITERATURE CITED
1. Hersman, L. E. BiodegrAdation of drilling fluids: Effects on groundwater chemistry. NNMSI Milestone Report. In preparation. 2. Montazer, P. and W. E. Wilson. 1984. Conceptual hydrologic model of flow in the unsaturated zone, Yucca Mountain, Nevada. USGS Water-Resources Investigations Report 84-4345. Lakewood, CO. 3. Seigal, B. Z. Science 1983, 219, 255. 4. wood, J. M. Chem. Scr. 1983, 21, 155. 5. Wood, J. M., Wang, H. K. Environ. Sci. Technol. 1983, 17, 582A. 6. Strandberg, G. W. 1982. Biosorption of Uranium, Radium and Cesium. In: "Impact of Applied Genetics in Polution Control". Eds. Kulpa, Irvine, and Sojka.
7. West, J. M., I. G. McKinley, and N. A. Chapman. 1982. Microbes in deep geological systems and their possible influence on radioactive waste disposal. In: Radioactive Waste Management and the Nuclear Fuel Cycle. 3(1) 1-5. 8. West, J. M., I. G. McKinley, and N. A. Chapman. 1982. The effect of microbial activity on the containment of radioactive waste in a deep geological repository. In: Scientific Basis for Radioactive Waste Management. 5. 9. Champ, D. R., W. F. Merritt, and J. L. Young. 1982. Potential for the rapid transport of plutonium in groundwater as demonstrated by core column studies. In: Scientific Basis for Radioactive Waste Management. 5. 10. Treen-Sears, M. E., B. Volesky, and R. J. Neufeld. 1984. Biotech. and Bioenqr. 27, 1323-1329.
13 11. Christofi, N., J. M. West, and J. C. Philp. 1985. The geomicrobiology of European mines relevant to radioactive waste disposal. British Geological
Survey. Report FLPU85-1. 12. Rice, T. R. and V. M. Willis. 1959. Linnol. and Oceanog. 4, 245-277. 13. Honda, Y., Y. Kimura, H. Kitada, and H. Fukumitsu. 1973. Uptake and accumulation of radionuclides in seawater by plankton. In: Annual Report Just. Atomic Power, Kinki Univ. 9, 19-24. 14. Strandberg, G. W., S. E. Stumate, and J. R. Parrott. 1981. Appl. and Environ. Microbiol. 41(1), 237-245. 15. Dreher, K. T. 1981. Removal of uranium and molybdenum from uranium mine wastewaters by algae. Masters Thesis, New Mexico Institute of Mining and
Technology, Socorro, NM. 16. Sakaguchi, T., T. Horikoshi, and A. Nakajima. 1978. J. Ferment. Technol. 56(6), 561-565. 17. Horikoshi, T., A. Nakajima, and Sakaguchi. 1978. Agric. Biol. Chem. 43(3), 617-623. 18. Aoyama, I., K. Inomo, and Y. Inoue. 1976. J. Radiat. Res. 17, 69-81. 19. Marchyulenene, E. D. P. 1978. Ekologiya. 2, 80-81. 20. Neilands, J. B. 1974. Microbial Iron Metabolism, Academic, New York, NY. 21. Raymond, K. N., G. Muller, and B. F. Matzanke. 1984. Complexation of iron by siderophores a review of their solution and structural chemistry and biological function. Topic in Current Chem. 123, 49-100. 22. Humphrey, B., Kjelleberg, S., Marshall, K. C. 1983. Appl. Environ. Microbiol. 45, 43.
14 e -
-~~O 2i0,.
fl-Ic
7MM=BY maErSKWU3MSM:
.CWEIrMC
Larry E. Hersmlan, LS-2
Los Alamos National Laboratory
¾
S.'..
¾ Co
;', - -
-rt a. .4,.... 'S. * $-Iz bS -. - S.. ... St Abstract
cogpounds produced by microorganisms Siderophores are lowmolecular-weight to solubilize the very insolulble These com.o are used by microorganisms of Pu4 # and Fe", it may be Fe 3 ÷ cation. Because of the atomic similarities Pu 4'. The chelation of Pud+ by possible that siderophores could bind .. . side-rophoresa increase its transport, and tre transport of other actinides, nuclear repository at Yucca Mountain. away from the candidate high-level in our laboratory and is the The binding of Pu" is being investigated indicate that the siderophore of this report. Preliminary results subject being to Pu4. Experiments are now by a Pseudommas 2. does bind pýduced complex. formation constant of the Puý*/siderophore performed to determine the studies now being. his info rationwi" e incorporated i• the sorption
performed at Los Alamos.
1.0 Introduction
by the Nevada Nuclear Waste stErage the process of characterization, "During site for a . the geology and hydrology of the Investigations (NaNSi)Project, of at Yucca Mountain, Nevada, high-level nuclear waste repository candidate radius of fluids were used. Within a 3-km millinn gallons of drilling several that Shaft Facilit." (ESF) it is estimated the proposed site of the Exploratory fluids were released into the block 15,000,000 gallons of drilling nearly these wells area. The drilling logs from (candidate site) or in the immediate the fluid was lost in the Topopah Spring indicate that a significant amount of of the repository. The Topopah which would be the geologic location Member, as a welded tuff with a lo. permeability Spring Member has been characterized Montazer and Wilson (1) have therefore and a high content of fractures. exceed downward lateral movement of water may suggested that in-this member of drilling* the recent discovery y, u, rates, a statemný pusataebwithout fluids). Interesting j fluids in-the USW Z-i hole (air-drilled holes (USW from the closest fluid-drilled is located-several, hundred meters zone,., at a depth .. SG-1 a H_ I)-, -and its drilling was limited to the, unsaturated the repository. approximately equal to that of
materials several other forms of organic in addition to those drilling fluids,
7 • i -J IMI
of th ...... will be intoduced Lntot.e bloc,-•of.thesexploraction hydraulic fluids, and shaft, items such as diesel fuels and exhausts, routinely. It can be anticipated additional drilling fluids will be discarded used during the construction of the that manry of the sam materials will be is sealed there will be ~"'"~ rpostoys wll ~refore, before the repository i;t., A comnprehensive ~'"in forganic material-i4at~Ja toAb usdUring the construction and inventory of the fluids that are going at LANL (2). operation of the ESF is now being prepared
and organic materials may be Our specific concern is that these fluids number of microorganisms, which in utilized as growth substrates by a large elements away from the turn may influence the transport of radioactive transport.in one or more of the repository. microorganisms can affect
following ways: chemistry through changes 1) alter the composition of the groundwater in pH or Eh, by sorption onto a nonmotile 2) retard the transport of the radionuclide solid phase, movement, 3) transport the radionuclide via biological via colloidal dispersion, . ) transport the radionuclide chelating agents. 5) solubilize radioactive elements bYpoducing to: study the chelation cf In this paper we report experiments designed actinide elements.
ecology ,is that microorganisms are A very important principle of microbial of.. ietalsthrough soils. one example able to strongly influence the movement powerful chelating agents called siderophores is the microbial production of the last three that are s,'&to solubilize the very inso,l .lFe i-cation.i In and characterized. They have "'decades over 80 siderophores have been.-isolated as 1052. At pH7 the equilibrium formation constants for Fel+ as high Fe. •isalppr=xiUmately) 10u M. For concentration for ferric ion (as free a total iron microorganisms such.as. enteric bacteria, growth, that concentration (10- M) t-. •.1iýfor•,t f_.chelating aget.su.ch as Sis-many-orders-of-magnitude too low. Only-.+pow.r the environment and facilitate its thL-siderophores can mobilize iron from transport into the microbial cell. ^- -2- as a-low-molecular-weight (500-1000. Neilands ) has. described a siderophore daltC) Virtually ferric-specific ligand, the biosynthesiS of which.is iron to the the function of which is to supply carefully regulated by iron and metabolism, microbes in several critical stages in cell.* As iron is involved affinity system its acquisition. The high evolved multiple'systems for have receptors'~ rophores and the matching membrane-associated is composed,' f the-side discussed in of the high affinity system is (4); only the- former component 3, Fe 2 to Fe -the concomitant oxidation of appearance of O0 in the atmosp=ere, (5). This explanation the latter as ferric hydroxide and the precipitation of in aerobic and facultative occurrence of siderophores accounts for the general anaerobic conditions) under both aerobic and anaerobic{(able'- to grow complexes in which the siderophores are octahedral microorganisms (6). Ferric 5 c cofi guration, and rapidily:'.p ,d me-tal. 'ion: is d! ,i ~eltron coordinate considered to be "hard" siderophore ligands are exchangeable. Generally for th" softer" acid cations, bases, and4Aherefore show little affinity
aneswof relea~eof i-cavia'a're tion step. including -Fe÷, providfig,,a extremely of the siderophores is' their "The sin•le umst outstanding feature ion (7). high affinity for the ferric
3 Puý" are similar in their that because Fe * and Neilands (3) believes Pu"+ may ratio (4.6 and 4.2 respectively), respective charge/ionic-radius 3 and coworkers (8) have to Fe+. In fact, Raymond possibly serve as an analog of siderophore-like tetracatecholate successfully synthesized a series as backbones. The both linear and cyclic tetramines ligands.'with catecholate satisfied by the four nature of Pu'÷ is well eight-coordinate therefore. binding of the Pu.* one cculd moieties, resulting in an effective approximately the same would bind to Pu4÷ with assume that a siderophore 3 of this information to as.,that with Fe ". The relevance formatio constant because waste repository is quitel's9nificant, the proposed high-level nuclear having a strong metabolic need-for' a large population of soil microorganisms the plus four oxidation-state actinides. iron could co-metabolically chelate * a large energy is'large enough (provided with If the bacterial population elements could be chelated, it is possible that radioactive -source)-v•'then the net actinide ment. eThrefore, in the solubilization of the resulting of the actinide an increase in the transport effect of chelation -would be -3- A purpose of the aqueous phase found at Yucca Poumtainl "elements through the via the potential for solubilization microbiology subtas 1 to.estimate as it applies to the significance of this effect chelation and to predict the : performance of the ,repository.
2.0 Materials and Methods
: K HPO , 6 g; experiments consisted f (g')1 2 4 The medium used for most of the acid, 4 g; pH 1 g; MgSO'3H2 O, 2 g; -succinic. Km•o,, 3 g; (NH4 ) 2 SOC, of the medium through by passing all of the constituents 7.0. Iron was removed Wra. For of iron was.adjusted to 1.0 Chelex 100 colum.. The concentration 0 :--a temperature (22 C), while cultures were incubated at room " large preparations, siderophore at 360C. The presence of tie smaller preparations were incubated ml FeCI in of an equal volume of Fe(C1O4 )3 (5.0 was detected by the addition at sample was then centrifuged of the spent medium. The 0.14 M HClO4 ) to i ml or in a nt indicated the 1800 g for 5inanadeeamberco presence of the siderophore..
a 24-h culture of a medium were inoculated with Three 2-L batzhes of succinate site) and incubated Pseudomonas sp. (previously isolated from the Nevada Test incubation the agitation. After 40 h of room temperature, under constant at the cell-free (4000 g, 30 min at 4C) and "cultures were centrifuged mL. The pressure to- approximately 100 was evaporated under reduced supernatant .of chlorofom:T extracted with an e4Y concentrated material was then was treatedwith an equal (1:1, v/v). The organic phase phenol solution was phase of this second partition of water and ether. The aqueous volume aqueous an equal volume of ether. The extracted two additional times with about 30 mL. This further concentrated to phase of the finalpartiti•-.was and a CM-Se~hadex.-C-25column aqueous phase was loaded onto concentrated 20 drops per acid (pH - 6.5; flow rate, eluted with. 0.1 M pyridine/acetic at _ a.^alo.rkae. was measured Ten-milliliter fractions were collected min). The fractions Beckman Instruments. Inc.). nm (Beckman Spectrcphotometer, 280 P2 column and further purified on a Bio-Gel containing the siderophore wc:e eluted with glass-distilled water. -4- - ______I
was utilized: Protein Assay..- A modification of Lowry's method 50 pL of 0.15% 1. sample + dd H120 was brought to 1 mL, to which was added deoxycolate and 100 pL of 72% TCA; mn at 40C; 2. it was centrifuged at 11,000 9 for 30 mixed well; 3. 1 L Of ddOIq was, adde to the esa incubated at room. 4. reagent A (1.0 nL)-was added, mixed well-'and temperatur for 10 min; incubated for 30 min; and 5. reagent B (0.5 nrL) was added, mixed well, 6. the absorbance was read at 750 run.
Reagent-"A - 1 vol copper-tartarate carbonate I vol 0.8'4 N NaC9 .. vol 10% SDS 1 vol dd H10
Reagenti3,B- 1 vol Folin (2N) 1vol dd 1ý20
the presence Se W.• remnce of siderophore was indicated by to Fe(CO ) ] of a deep a••r color upon the ^ddition of FeCl 3 (later changed 4 3 the uninoculated controls no color change was -to the spent medium. For the presence of a observed. :This-'deep amber color also suggests because Z catecholate-type, hydroxamate-type siderophore, as opposed to a addition of ferric iron to spent according to Neilands (3) and Emery (9), upon are-wine "medium, trishydrokamates are orange colored, while triscatacholates "colored.
peaks collected from the Siderophore Isolation and Purification. Three rim. The first and third peaks CM-Sephadex.C-45dolumn were observed at 280 while the u sual-.-c ee- d"edisplaying-a-deep color chainge The fractions containing the second peak showed less of a color change.- test tubes. siderophore were freeze-dried and stored in sealedplastic
Approximately 1 mg High-Pressure Liquid Chroontography (HPLC) Purification. CM-Sephadex C-25 fractions of freeze-dried siderophore from each of the three and injected into a waters -.Ve reconstituted in 200 pL of HPLC-grade water
:-: • "' ,: ... :•- -5- A:.:
7; (flow rate, 2 mW Mi; model 590 tlPLC.equipped with.a C-18 column The solvent gradient was min of .. attenuation, 1.0; absorbance, 206 nm). from 100% solvent A (100% H20) isocratic flow (10), and 1-h linear gradient, For the first of the Sephadex to 100% solvent B (50% ;0, 50% acetonitrile). approximately 2.0 and 24-45 min (Fig fraction, major peaks were detected at four major HPLC peaks at la). The second Sepadex fraction contained min (Fig. lb). The third Sephadex approximately 2.5, 6.0, 14.0, and 22.0 4.0 min (Fig ic ). EEach of these peaks fraction contained only one peak at FeC! [has not been 14Q been tested for siderophore content using the 3 and third Sephadex fractions most using Fe(ClO4 ),)], and for the first repeated peak early (2.0 and 4.0 min, respectively) of the siderophore was found in the was detected in the second Sephadex (Fig. 2a and 2b). Very little siderophore fraction
to evaluate the signif.;-ance of Formation Constant with 29 u. In order the formation constant of the chelation, it is necessary to determine we prefer to work at neutral pH, in siderophbre/actinide complex. Ideally conditions found at Yucca order to remain consistent with environmental complexation at Mountain. For ... Pu•"the best way-to-avoid-spontaneous as 10-s M or lower. work with very dilute solutions, such 4 neutrality is to an acidic 29Pu have been prepared by diluting Classically these solutions using carbonate complexation to feed solution. Hobart (10) is currently actinide. Regardless of the preparation stabilize the oxidation state of the to avoid ccaplexation. However, method, the plutonium ion must be diluted classical spectrophotometric and because of the low actinide concentration, 39 Pu'* complexation cannot be used to electrocherical analysis of siderophore the complex. determine the formation constant of
between the chose to use competition experiments For that reason we to meas formatiort-constants. siderophore and a known tmeal-chelating agent- resin which shows unusually high Chelex 100 is a chelating ion exchange heavy metals. It is a styrene-divinyl preference for copper, iron, and other acid residues are attached, benzene-copolymer matrix to.,which iminodiacetic to that of iminodiacelic acid. hence its selectivity for metals corresponds Fe 3 and described the atomic similarities of Raymond and coworkers (8) ha'e Pu 4 Chelex 100 will bind to appropriate to assime that Pu 4. It is therefore
-6- 3 "'and therefore can be used to determine in a mannez analogous to that of Fe and p4.P3• the formation constant between siderophore
feasibility of using Chelex 100 Experiments were first,.performed to test the and experiments., Cc~etition~,among Chelex'100 in com~petition The (EDIA) for ferric iron was tested. ""-^-- ethylbnediaminetetraaceticacid as follows: preparation of the experiment was EDTA solutionsi 1) made up 3 mL total volume of seven different 0.5, 0.2, 0.1. .05, and .02 M EDTA 2) transferred 0.2 mL of 2.0. 1.0, into 3.8 mL of water; in half; 3) dividd the,4.0 mL.-volume :of each Chelex 100 sediment Ichelex beads-" 4) to one-half added mL. of saturated saturated with Fe(CIO,)3]; and of saturated Chelex 100 w;*ter 5) to the other half added I mL (supernatant).
I 15-mL of W2 in each of the reaction vessels The final concentrations x 6.G7 x 10-2 uM, 3.33 x 10". pM, 1.67 plastic, Pyrex centrifuge tubes) were YM, 1.67 x l0- xiM, and 6.67 x 10 " M. 10-2 gM, 6.67 x 10-3 JM, 3.33 x 10-3 for 10 h at room temperature. Two The centrifuc-- tubes were agitated and adsorbance was, measured on milliters of :fluid~was. removdzfrom each tube, spectrophotometer, using the corresponding a Perkin-Elmer model 552 scanning performed as a blank. This experiment was saturated chelex water supernatant presented in Fig. 3. in triplicate and the results are
The using siderophore instead of EDTA. A similar experiment was performed compete with to determine if siderophore would purpose of this experiment was adjusted to concentration of siderophore was Chelex 100 for ferric iron. The the moleculage.ieght of 0.014 M, based•upw-the-assumption that approyimatelY direct 1000 daltons. Although we have no the siderophore is approximately is that. value.,rthis,- evidence that the ýie&dar--n._-ight-of the s iderophore• °n using molecular sieves. The approximation was based on.sizei while being retained by a 500 siderophore passed through a 2000 sieve, either 5.0, 25, 125, sieve. The volume of chelex added was molecular-weight in results of this experiment are presented 625, or 1000 yL (bed volume). The
-7- Si p Fig. 4.
the feasibility of using Experiments were then performed to determine 3 One milligram of siderophore was cpetition experiments with 2 Pu'- Solutions of 5, •2reconstituted in 2--L oMf triple-distilled deionized water. which of triple-distilled deionized water 50, or 500 pL were added to 10 mL Chele4O0-'beads (Bio-a)- having an contained 50oL of washed, deinized 0.1 mL of approximately of 0.4 meq mL- To these solutions exchange capacity and, -ixed for 24 h at room . .0-' tO i0- n "PU feed solution was added removed by centrifugation and 5 mL of temperature. The Chelex 100 beads were mL of scintillation vials, to which 5 supernatant was placed in liquid and 10 mL of Insta Gel scintillation filtered J-13 water (0.05 Nucle•pore) containing both siderophore and were added. Activity in those tubes flour Chelex in those tubes containing only Chelex 100 was compared with activity
- 100.
3.0 Results and Discussion
for over 80 Although siderophores have been isolated - . siderophore Assay. is known about these compounds. This lack species of microorganisms, little throughout all phases of siderophore of information tends to be uniform by the tNWSI microbiology subtask "research. Therefore, much of the work done we found that a procedure, or the development of procedures.- often "has been results. The use of FeCl 3 in the part of a procedure, yielded conflicting Sometimes a fraction (from the C-25 assay procedure yielded such results. siderophore would test positive was suspected of containing the column) that g•_ • oieohoews the-o . At first it t th sometimesfit would:test nega upon the addition of FeC)., or presefl.tat, flwtnegti assumed that the sideroprwanolt-really' was growth of the procedure wasae fauilt (e.g., fractions and that some other part it determined that -" - conditions). However, after extensive experimentatior :media. components reaction of FeCl 3 with other the problem was the nonspecific thereby interfering with the that often resulted in flocculation, Alternative compounds such as spectrophotometric measuremEnts. ) 9 H O, and )2'(S04 )" 6H.0, Fe, (SO1)3'xH 2 O, Fe(NO 3 2 2 Fe(NH 4 )2 C6 H7O ), Fe03, Fe(NH4 -8 .1.••' 7
assay. Ferric perchlorate Fe(C10 ) were all tested for use in the 4 3 was used in to give the best results and therefore jFe(ClO4 )3 ] was determined subsequent assays. intrOdGx .o, the production of -•-86nientrat• of'•Wr • As stated inthe
regulated by the presence of iron. siderophore by microorganisms is strictly siderophore by reducing the amount of Normally one induces the production of is a requirement one has to be careful, because iron available iron; however, i then microbial growh c•an becc• for growth, and if ron.beco•es limiting siderophore production could bec impaired. If thathappens then overall maximum bacterial growth prior to..., reduced-. e' "e:i" fore,-! is to achieve both of which should occur before achieving maximum sideorphore production, other words, the cells should be cells reach stationary phase. In the log of iron that allows them to enter supplied with an initial concentration log growth phase, the concentration phase. once the cells have entered -growth of sideropore of iron becometslimiting and results in the induction order to achieve these results, a maximu number of cells. In production in a from the growth medium and then add one must first remove all of the iron known concentration-of iron.
ssing stock solutions of each of the The removal of iron was a ~c9she-by through a column of Chelex 100 the water, used in the growth medium salts, and to of simple experiments were performed ion exchange beads. Then a series in the greatest production "determine what concentration of added iron resulted obtain to iron-free growth medium to "of siderophore. Iron was added concentrations of either 0.1, 1.0., 0,"-°r-lO0", results varied among the experiments, repeated six times. Even though the concentration of iron most often yielded those flasks containing the 1.0-yM siderophore. Therefore,was adjusted-to~l.0,, in subsequent -..- •-•...... " ." .. ,: •,-••~irlthe highest. concentration'K ofentration of--iron
became immediately obvious that the HPLC Analysis. Using HPLC analysis it there was more than one siderophore siderophore was either not pure or that from the C-25 Sephadex colmrn (Figs. present in each of the peaks collected la, ib, 1c).
-' . . -9-
: . m 3' -- : purification have been analyzed b- :the-. HPi "Three ditferent C-25 column peaks
fractions that tested positive with andsjnseachycase it was the early method,. hn n -5Sephde (Figs. 2a, -2b). It is unclejar why moe the iron assay the iron: assay. Perhaps the f raction tested. positive, for. siderophore with Ps.... -u.-.,aSA2* wa producing-ore-wo••ne mornge. erhaps components., siderophore was breaking down into several
to produce more than one siderophore It is not uncommon for a microorganism to produce pyochelin and pyoverdin, Pseudomonas aeruqinosa is known (11). aerobactin, to produce both enterocholin and Escherichia coli has been found diyroxybenzoie'acid, azotochelin, and v • A--zoto1icter vinelandiiLproduces and is controlled in a sequential manner, azotobactin. Siderophore synthesis of iron limitation (12). Organisms is governed by the increasing severity expending energy synthesizing siderophores, appear to conserve energy while but most effective, siderophore only for the synthesis of the most complex, - of the However,ý, in a receni investigation when it is essential to do so. fluorescens it was and ferribactin by Pseudomnas production of pyroverdin P late in the culture, even though that ferribactin was produced found 14, 15). most, effective o6f -the two (13, pyoverdiný, was thought to be the demand is not known to synthesis dependent upon iron Therefore, sequential that all of these examples it is believed for these siderophores, but in exist is critical because one of the siderophores multiple siderophores are produced information, it deprivation. Based upon this for growth under severe iron the Pseudomonas sp. used in our studies "-S. would not be surprising to find that siderophore. may be producing more than one
C-25 Sephadex peaks is that possible explanation of the multiple A second that we could be occurring. The microorganism degradation of the siderophore sp., isolated from the NTS, with is an unknown Pseudomonas have been working toit sli,.....co..d. and coeYk'-ea P. stutzeri, Due. -t..04 .....0i ' e a. o. enique,--inc..- .i...."zi-d- fy siderophor- ...... "assume ...... that ...... many ...... of its m WM Becaulse,-.. "!•:!•'.sot.f.. siderophore. The meth " s t.n -h-iteaue been modifications of procedures taken from. have sp., it is specifically for this Pseudomonas these methods were not developed method, may be causing a partial that a method, or part of the possible improving As a precaution we are continually degradation of the siderophore. I-10-. - " "•o .• • ;. I
,. , . . . .', . ..
• •e,,-- "" " " ". - :-• : i•40 this!the.e prprocedu• ceduis-Oý, •ie-•.resig - theI'.iterature.... for better iethods
chelation research project Formation Constant:.. one of. the objectives of the between the siderophore and actinide is to determine the formation .constant obtained, then we can eaea m ore elements. ""ce:this:information is in ,t of meaningful assessmentoF t-hole that microorganisms rock. Preliminary experiments were actinide elements.throtigh tuffaceous of using competition between the ligand designed to determine the feasibility and Chelex 100 for the metal ion.
for divalent cations, EA is a chelating ligand having a strong preference as seen in Fig. 3. Chelex 100 for ferric iron, 3 and it clearly cu¢mpeted with EDI F e * the adsorbance of the concentrations of EDTh, with increasing 3 the EDTA was removing the Fe * ion complex increased significantly. Evidently _ frcm the saturated Chelex 100 resin.
2 trend. The results of"':the •sideroI e competition experiments show a similiar of the Chelex 100, the adsorbance maxim-n With increasing..concentrations of (Fig. 4). Therefore, we can siderophore/Fe•- "ex significantly'decreased the ferric ion fromt e si derophore. assume that the Chelex 100 was removing was out that in this experiment the iron However, it is important to point the iron was and not to the Chelex 100. When added to the sider&ohore first no as was done in the EDTA experiment, added to the Chelex 100 first, Yet, when the iron and siderophore/iron adsorption could be observed. possible to observe a decrease in the siderophore were mixed together, it was Obviously the Chelex 100 was able to siderophore/Fe'÷ complex adsorbance. to but the siderophore was unable remove ferric iron from the siderophore, to explain 100. At this time we are unable remove ferric iron from the Chelex this anomaly.
that competition between __ of preliminary'experiments demonstrated R The final set prove to be useful technique. sideopre a-nd39Chel4xl00£or wP may and actinide elements'?4 calculating-the. b-t'--'SiderOPhore d".to\$ntn only once, and crude siderophore Although this experiment was performed that at a higher amount of preparations were used, the re'sults demonstrated 4 siderophore. It appears that the Chelex 100 less 23 Pu was bound to the -11- 1,ýk 4 thelformation constant between siderophure does bind to 2'Pu * and that 4 Chelex 100 and 2'Pu*. siderophore and 239Pu " is similar to that of
" ...... 4.0 Conclusions ......
is very interesting. An unknown The progress of the chelation experiments been found to strongly bind to microorganism, isolated from the NTS, has brigians 239pu4,. This species is krg,--, topodc -pwrfullmt- have been-isolated and are now being called siderophores. These siderophores experiments suggest that the purified in the laboratory. preliminary are :now being conducted to siderophore does bind to 23Pu4-. Experiments ccoulex. The determine the formation constant of the siderophore/-3,-PUG+ will be used to predict the information generated from these experiments the tuffaceous. rock foL-ud at Yucca S..m.• nt of actinide elements through This information will be Mountain, as influenced by microorganisms. now beinguperformed at Los A1amos.. incorporated into the sorption studies
5.0 Literature Cited
1984. :USGSMWater-Resources 1. Montazer, P., and W. E. Wilson. "investigations Report 84-4345. Lakewood, CO.
7" 2. Wes:, K. 1987ý NNWSI Milestone report 1162.2
50: 715-731. 3. Neilands, J. B. 1981. Ann. Rev. Siochem.
Nutr.. 1:'27-,46.-_' 4. Neilands, J. B. 1981. Amn. Rev.
'11:731-740.- 5. Biedermann, G. and P. Schindler. 1957.; ti ?Sc
Berlin. 11: 45-70. 6. Neilands, J. B. 1972. Struct. Binding.
1979. Acc. 0=hem. PRes. 12: 183-190. 7. Ray-mnd, K. N., and C. j. Carrano.
. • - ...... 2,- mI
o _-.... _ I
8. Raymond, K. N., G. Muller, and B. F. matzanke. 1984. Curr. Chem. 123: 49-100.
9. Emery, T. 1971. Adv. Enzymol. 35:1354185. in. 10. Hobart, D. E., P. D. Palmer, and T. W. Newton. 1985. Annual Repor NNWSI. 92-95.
I,-- 11. Ankenbauer, R., S. Spiyosacjati, ard C. D. CoX. 1985. Infect. 49: 132-140.
496-502. 12. g W1 1 - 3yer. Bacteri•l. 158:
Microbiol. 107: "13. Meyer, j. p., and M. A. Abdallah. 1978. J. Gen. 319-328.
5. 14. PhilsonS. B., and K. Llinas. 1982. 3. Biol. dhem. 257:8081-808' 90. 257: 8086-80 15. Philso , S. B., and m. Llinas. 1982. J. Biol. Chem.
Figures
a Sephadex column. Figure la. HPLC chromatograph of fraction 17-1 taken from
Sephadex column. Figurelbz,ýHPLCA chrcmatograph of fraction 17-2 taken from a
Sephadex column. Figure 1C.ý --HPLCachromatograph of fraction 17-3 taken from a
• Figure 2a. Adsorption maxinnms of fractions 1 and 2-[plus-Fe(ClO )3--taken from HPLC run 17-1 (figure la).
for ..... re. Figure 2b. Analysis of fraction from ...... -r
-A S.,,i:..- . '-..T -•-: . :•.. .• - -..: .- -.•.• g . -. .: "---. ••::-:!i! :•• ...." -.- L
in the presence of 3- 3.Adsorption (280 rm) vs WIA concentration I;~;;ziK>,Figure ele x 100.[p,"ksjb1. presence of siderophore (plus I ".....- re 4. Adsorption,(295 im) vs cdelex in the I p
I - -
-V
rY.::j- :
"M,m! 77
S...... ,:•.k,.,:• ~i:-: . .... •• ....e _.. .• -14- FIG. IA
1.0
0.9
0.8
*
p.-.
L * 0.6
a 0.5 0 < 0) <• 0.4
0.3
0.2-
0.1
5-. 0 5 10 15.20 25 30 35 40 45 50 TIME (min)
FIG. 'IC
* ' *-'t cZ4X; %. FIG. ic
0.9
0.8
LU 0.6 0 z c a 0.5 Zr
< 0.4
- I -- 1
'0.1
-1
0 5 10 15 20 25 30 35 40 45 TIME (min)
-17-
3 . . - - IM1417½WN-t TM-.r.
FIG. 2A
4 -r-
c-a0
A-.-C cc,
200 300 400 WAVELENGTH (mm)r . . .. . - " 4 ...... ,•,. •., • -. ...s
w z (50 0 (I) 42
300 400 •,•••WAV ELE NGTI± ,..
.,...5 ....
•~.L,_j
2 .
°€.o
-19- -s-.,
4 4 A~~1,1 e isr-4
cl%
- I
1.0 x 1 .3 CHELEX [ ]
Cal. I1i tc1
TRANSPORT BY MICROORGMISMS:
COLLOIDS
Larry E. Hersman, LS-2
Los Alamos National Laboratory Abstract
Colloids are suspected to play an important role in the dispersion of toxic wastes, heavy metal contaminants, and radioactive wastes in the subsurface environment. Preliminary studies performed at Los Alamos National Laboratory suggest that the presence of microorganisms significantly accelerates the formation of colloidal agglomerates. Within 50 hours, nearly all single colloids had formed multi-particle agglomerates. The experiments presented describe the role that microorganisms play in the formation of colloidal agglomerates and how this interaction affects the transport of radioactive wastes.
1.0 Introduction
During the process of characterization, by the Nevada Nuclear Waste Storage Investigations (NNWSI) Project, of the geology and hydrology of the site for a candidate high-level nuclear waste repository at Yucca Mountain, Nevada, several million gallons of drilling fluids were used. Within a 3-km radius of the exploratory shaft it is estimated that nearly 15,000,000 gallons of drilling fluids were released into the block (candidate site) or in the immediate area.
The drilling logs from these wells indicate that a significant amount of the fluid was lost in the Topopah Spring Member, which would be the geologic location of the repository. The Topopah Spring Member has been characterized as a welded tuff with a low permeability and a high content of fractures. Montazer and Wilson (1) have therefore suggested that in this member lateral movement of water may exceed downward rates, a statement that was substantiated by the recent discovery of drilling fluids in the USW UZ-1 hole (air-drilled without fluids). Interestingly, UZ-1 is located several hundred meters from the closest fluid-drilled holes (USW G-1 and H-l), and its drilling was limited to the unsaturated zone, at a depth approximately equal to that of the repository. In addition to those drilling fluids, several other forms of organic materials will be introduced into the block. During the construction of the Exploratory Shaft Facility (ESF), items such as diesel fuels and exhausts, hydraulic fluids, and additional drilling fluids will be
-I- discarded routinely. It can be anticipated that many of the same materials will be used during the construction of the repository as well. Therefore, before the repository is sealed there will be a significant amount of organic material located near it. A comprehensive inventory of the fluids that are going to be used during the construction and operation of the ESF is now being prepared at LANL (2).
Our specific concern is that these fluids and organic materials may be utilized as growth substrates by a large number of microorganisms, which in turn may influence the transport of radioactive elements away from the repository. Microorganisms can affect transport in one or more of the following ways: 1) alter the composition of the groundwater chemistry through changes in pH or Eh, 2) retard the transport of the radionuclide by sorption onto a nonmotile solid phase, 3) transport the radionuclide by biological movement, 4) transport the radionuclide by colloidal dispersion, 5) solullize radioactive elements by producing chelating agents. In this paper the progress of experiments designed to study colloidal dispersion will be discussed.
Colloidal dispersion has been implicated as a means of transporting toxic wastes, heavy metals, and radioactive wastes through soil and rock systems (3; J. McCarthy, Oak Ridge National Laboratory, personal communication). When attached to a colloid, these substances are unable to participate in adsorption/desorption reactions with the soil or rock matrix. These substances then move at an accelerated rate with the colloids though the soil or rock matrix. If, however, colloids become attached to one another to form agglomerates, then these particles would no longer be available to participate in colloidal dispersion processes, because of size exclusion. Obviously, with increased agglomerate size, the effect of size exclusion would also increase, and the net result would be an overall decrease in the transport of radioactive wastes. With that supposition we investigated the influence of bacteria upon the agglomeration of clay colloids.
-2- It would be worthwhile to first review what is known regarding the interactions between microorganisms and colloidal particles. Those interactions include the attraction processes, adhesion, adsorption, the effects of colloids on microbial metabolism, and flocculation.
1.1 Attraction of Bacteria to Solid Surfaces
Since many natural habitats have a low nutrient status, solid surfaces are potential sites for concentration of nutrients (as ions and macromolecules) and, consequently, of intensified microbial activity. The movement of water across a surface provides increased opportunities for microorganisms to approach solid-liquid interfaces. In addition, there are many physicochemical and biological attraction mechanisms operative in the inmmediate vicinity of interfaces: 1) Chemotaxis. Mobile bacteria are capable of a chemotactic response to low concentrations of nutrients introduced into a normally nutrient deficient system. Chemotaxis, of course, cannot account for the attraction of nonmotile bacteria to solid-liquid or other interfaces. 2) Brownian motion. Colloidal particles are in a state of continual random motion (Brownian motion) caused by the chaotic thermal motion of molecules that collide with each other and with particles suspended in the liquid; 3) Electrostatic attraction. The interaction of negatively charged surfaceý of bacteria with solid surfaces probably depends on the properties of the surface in question. Because most surfaces in nature are negatively charged, it is unlikely that electrostatic phenomena are involved directly in the attraction of bacteria to such surfaces. It should be emphasized, however, that solids in natural environments often acquire different surface properties through
-3- sorption of macromolecule compounds to the surface. Therefore, the surface, as "seen" by the bacteria, may be very different from the original surface. 4) Electrical double layer effects. When two negatively charged bodies are in close association, they may be repelled from or attracted to each other, an effect that depends on the thickness of the interact ing electrical double layers, which, in turn, is dependent on the concentration and the valency of the electrolyte. 5) Cell-surface hydrophobicity. It is quite reasonable to assume that part or all of the outer surface of some bacteria is hydrophobic. It is therefore reasonable to consider that such bacteria are rejected from the aqueous phase and attracted towards any nonaqueous phase, including solid surfaces.
1.2 Adhesion
The adhesion of bacteria to inanimate surfaces is widely recognized as having enormous ecological significance. Adhesion of microorganisms is involved in certain diseases of humans and animals, in dental plaque formation, in industrial processes, in fouling of man-made surfaces, and in syntrophic and other community interactions between microorganisms in natural habitats.
Most aquatic bacteria appear to adhere to surfaces by means of surface polymers, including lipopolysaccharides, extracellular polymers and capsules, pili, fimbriae, flagella, and more specialized structures such as appendages and prosthecae. Even though these surface components play a role in the initial, reversible adhesion, they often serve to anchor the bacterium at an interface by polymeric bridging.
The composition and quantity of bacterial cell surface polymers vary considerably and are strongly influenced by growth and environmental conditions. Although extracellular polysaccharides have been reported as being responsible for irreversible adhesion, this is not always true. For example, Brown et al. (4) demonstrated adhesion in mixed, carbon-limited populations despite no evidence of extracellular polymer production. Also, a
-4- nitrogen-limited culture resulted in poor adhesion, although large extra cellular polymer production was observed. It should always be kept in mind that polymers present between the cell and the substratum, but not observed in light or scanning electron microscopy, could be responsible for the adhesion.
It appears that, with respect to inanimate surfaces, there is a subtle balance between cell surface components that may reduce [extracellular poly saccharides, lipopolysaccharides (LPS)] or promote (fimbriae) adhesion to inanimate surfaces. Jonsson and Wadstrom (5) recently demonstrated that an encapsulated Staphylococcus aureus strain did not bind to hydrophobic Octyl Sepharose gel, whereas a noncapsulated variant showed binding capabilities. In fact, polyanionic extracellular carbohydrate material may not be primarily concerned with the initial adhesion processes, but rather with development of subsequent bacterial film (6). The presence of LPS reduces cell surface hydrophobicity and decreases adhesion to the air-water interface of Salmonella typhimurium (7).
At short distances, many forces are important (ionic and dipolar, H-bonds, and hydrophobic interactions). Therefore, it is possible that the variabilities in bacterial surfaces result in various types of interactions that occur simultaneously with a single bacterial type. For example, hydrophobic interactions adjacent to ionic or hydrogen bonds can stabilize an otherwise energetically weak binding complex (8). Doyle et al. (8) propose that adhesion can be described in terms of positive cooperability; i.e., once the initial bacterium is bound, compounds on the substratum may change conformation, creating new receptor sites for other cells. The formation of new sites could be the result of the influence of hydrophobic interactions, but enzymatic action exposing saccharide receptors or cell-cell binding at the high bacterial concentrations used should also be considered. Unfortunately, then, theories on specificity in adhesion at inanimate surfaces and the ecological significance thereof must be discussed in view of seemingly opposing experimental data.
1.3 Adsorption of Colloidal Clays
The adsorption of colloidal clays to bacterial surfaces has been studied
-5- by Lahav (9) and Marshall (10-12). Lahav (9) suggested that clay platelets may be oriented in a number of ways at the bacterial surface, as a consequence of the net negative charge on clay platelets and the existence of some positive charges on broken edges of platelets. Marshall (10,11) reported that species of Rhizobium with a carboxyl-type ionogenic surface sorb more Na÷ illite per cell than do species with a carboxyl-amino ionogenic surface. Using sodium hexametaphosphate (HMP) to suppress positive charges on platelet edges, Marshall (12) found that the HMP-clay did not sorb to carboxyl-type bacteria, whereas a limited amount of this clay sorbed to carboxyl-amino-type bacteria. He interpreted these results in terms of the electrostatic attraction between the platelets and the bacterial cell surfaces. Normal sodium montmorillonite particles sorb in an edge-to-face manner to carboxyl type bacterial surfaces, with positively charged edges of the clay attracted to the negatively charged bacterial surface. Sorption in this manner is prevented by neutralization of positive edge charges of the clay by HMP. In addition to a predominantly edge-to-face association at the surface of the carboxyl-amino type-bacteria, a limited amount of face-to-face association between the negatively charged face of clay platelets and positively charged amino groups was postulated. This face-to-face association is not prevented by neutralization of the edge positive charges by HMP.
1.4 Effect of Soil Particles on Microbial Metabolism
Adsorption of organic substrates on soils depends on the nature of the particulate matter, the organization of the fabric, the clay types, and the cation status of the soils, as well as on the concentration and molecular structure of the substrate. Therefore, the availability of substrates to soil microorganisms may be enhanced or reduced by the presence of particulates.
Sugars sorb poorly to clays (13). Metabolism of glucose in soil is inversely related to the degree of bacterial sorption, which is related to the participating bacterial species (14) and the soil cation status (15). Stotzky (16,17) and Stotzky and Rem (18,19) determined that kaolinite had little effect on bacterial respiration with glucose as a substrate, whereas montmorillonite significantly stimulated respiration. Novakova (20,21) showed that the sodium and calcium forms of a montmorillonitic clay stimulated
-6- glucose decomposition, but that these forms of kaolinite were inhibitory. Stotzky (16) also reported that when samples of montmorillonite were made homoionic to a range of cations, there was increased stimulation of bacterial respiration with saturating cation in the order Na >Ca >Mg >K >H. In a separate study, Stotzky (17) reported that bacterial respiratory activity was also related to the cation-exchange capacity and surface area of clays, but not to their particle size.
Many other excellent studies have been performed to determine the effects of clays on the decomposition of starch (22,23), aldehydes (24), pesticides (25,26), and protein (22,27,28). In most of these studies, the effect of the clay depended upon the clay type and the ionic nature of the solvent. In fact, these parameters appear to dominate the effects of clays on microbial activity.
1.5 Microbial Flocculation of Clays
In recent years several processes have been patented for the flocculation of clays, particularly clays derived from phosphate beneficiation. The use of microbial polysaccharides from such organisms as Pullularia, Xanthomonas, Arthrobacter, Cryptococcus, Hansenula, and Plectania was disclosed to flocculate finely divided inorganic solids in an aqueous medium (29). In another patent (30), the use of alkaline-treated microbial nucleoprotein is described for flocculating organic and inorganic wastes. Used in this process were nucleoproteins from the microbes Polangium, Myxococcus, Sorangium, Flavobacterium, Leuconostoc, Micrococcus, and Alcaligenes. Nucleoprotein derived from these organisms was treated with any one of a variety of alkaline ammonium compounds, including Ca(OH) 2 , KOH, NaOH, NH3 , Na3 PO4 , and quaternary compounds, which would raise the pH to the point where the microbial material would lyse and form a sol. Flocculation of suspended waste resulted when the concentration of alkaline-treated microbial material was present in concentrations of 1 to 500 ppm (30).
Floc deterioration can result from biological factors as well as physical factors. Synthetic and natural polymers used for flocculation of colloids may be subject to degradation by microorganisms, which could result in floc
-7- destabilization (31,32).
As can be seen from the above discussions, there are several ways in which bacteria and clay particles may interact, including attraction processes, adhesion, adsorption, the effects of colloids on microbial metabolism, and flocculation. It is entirely possible that such interactions could affect the distribution of individual particles in solution. A very simple experiment was performed in the laboratory to investigate the interaction of bacterial and clay particles and its effect on clay particle distribution. The primary focus of the experiment was to determine if bacteria are able to influence significantly the agglomeration rate of colloidal particles.
2.0 Materials and Methods
Sterile Wyoming bentonite clay (0.05 g) was added to 100 ml of sterile water. The suspension was mixed, and 1.0 ml of the suspension was added to 20 ml of nutrient broth. Three flasks containing the 20-m. nutrient broth/colloidal suspension were inoculated with a Pseudomonas sp., three of the same flasks remained uninoculated, and three flasks void of colloids were inoculated with the same bacteria. One flask void of colloids remained uninoculated, serving as a sterile control. With an Olympus Vanox microscope, these various suspensions were monitored for over 200 hours. Individual colloids, and agglomerates (clusters) containing 2 to 5, 6 to 10, 10 to 25, and >25 particles, were counted. Counts were made with a Petroff-Hausser bacteria counting chamber having a volume of 1/400 rmm2 by 1/50 mm deep, or 5 x 10-5 mm3 , or 5 x 10-8 ml. This experiment was repeated three times; the results are presented in Figures 1-8 as counts per 5 x 10-a ml.
3.0 Results and Discussion
These results clearly demonstrate that the presence of bacteria significantly influenced the rate of colloidal agglomeration. In the presence
-8- of bacteria, the number of individual clay particles decreased rapidly with time (Figure 1). However, in the absence of bacteria (sterile) (Figure 6), the number of individual clay particles increased with time, presumably because of the disruption of preformed agglomerates present at time 0. Both the 2- to 5- and 6- to 10-particle clusters tended to increase with time in the presence of bacteria, while the 10- to 25- and >25-particle clusters followed a more complicated function, first increasing and then decreasing with time.
Sterile clay suspensions followed a more predictable pattern. Generally, the preformed cluster groups (both 2 to 5 and 6 to 10) tended to disassociate with time, as indicated by the increase in single particles (Figure 6). The inoculated, noncolloid flasks displayed a fairly typical growth curve, and therefore the results are not presented. The sterile control remained sterile throughout each of the experiments.
From the results, one can clearly see that the presence of bacteria profoundly affected the distribution of particles in suspension. These results are extremely important to the NNWSI Project. As previously mentioned colloidal dispersion has been implicated in the transport of radioactive wastes in soil systems. Microorganisms (both indigenous and exogenous) will exist in the vicinity of the candidate site of the high-level nuclear waste repository. The results of this study suggest that an interaction will occur between these bacteria and colloidal particles. As a consequence, colloidal dispersion of actinide elements could be influenced by the agglomeration of colloidal particles.
4.0 Future Accomplishments
This laboratory experiment demonstrated that there exists a strong potential for microorganisms to affect colloidal particle distribution. However, it was not quantitative in design and therefore can only suggest the possibility of strong interactions between microorganisms and colloids. It is necessary to describe the mechanism(s) by which microorganisms influence the
-9- agglomeration of colloidal particles. Although the literature contains numerous studies that discuss the interactions of bacteria and colloids, none of the studies address colloidal agglomeration, as affected by bacteria. Hence, there exists little information regarding (1) agglomeration rate (2) agglomerate stability and (3) particle distribution within the agglomerate. The approach of future research is to conduct a series of experiments addressing each of these issues.
4.1 Agglomeration Rate
A key to understanding the bacterial influence of colloidal agglomeration is the determination of agglomerate formation kinetics. Although the preliminary experiments demonstrated the involvement of bacteria in the agglomeration process, they provided no kinetic information. Additional experiments need to be performed to establish the rate of agglomerate formation. This information would be very useful in understanding the types of attractions that are occurring between the clay and bacterial particles. For example, agglomerate formation occurring parallel to bacterial growth would suggest that agglomeration is simply a function of the number of bacterial particles. If, however, agglomeration is delayed until stationary growth phase, then one would suspect that agglomeration is "threshold" in nature and depended upon some other parameter, such as pH or the concentration of extracellular metabolic products. In a related issue, it would also be useful to determine if a threshold concentration of clay particles is needed to initiate aggregate formation. Only one clay concentration was used in the preliminary experiments; however, in the proposed experiments, the concentration of clay particles will be varied.
4.2 Agglomerate Stability
Another important aspect of the proposed research is to study the stability of the bacterial/clay agglomerate. From the preliminary studies one can observe that the number of particles within an agglomerate cluster changes with time. It appears that the large clusters are forming early, followed by a breakdown into the smaller cluster units (2 to 5 and 6 to 10); however,
-10- more detailed studies need to be performed to determine the interaction among these cluster units. In addition, the long-term stability of the clusters should be studied. Such studies should address the eventual fate of the clusters: is there one particular cluster unit that will dominate? Will there be an equal distribution of particles among all the cluster units or will all the clusters eventually break down to individual particles?
4.3 Particle Distribution Within the Agglomerate
The relative distribution of bacteria and clay particles within a single agglomerate should be determined. It is not known whether the ratio of bacteria to clay is uniform among all the cluster units, or if this ratio changes with the size of the cluster. It may be that single bacterium initiates cluster formation, or several bacteria in close proximity to one another may be responsible for aggregate formation.
4.4 Experimental Procedure
As in the preliminary studies, light microscopy will be used to analyze the colloid/bacteria interactions. An Olympus Vanox Research microscope equipped with two 35-amm cameras and one 4x5 polaroid camera will be used for the analyses. For most of the observations, either phase contrast or epi fluorescence microscopy will be used. In addition, the microscope will be equipped with a Sanyo VDC 3800 video camera having a digital analog converter. Image analysis will be done by a PC Vision Frame Graber coupled with an IBM-PC using an appropriate software program. Particle and cluster analysis can therefore be performed much more quickly than was done in the preliminary studies, and data storage will be automated.
5.0 References
1. Montazer, P., and W. E. Wilson. (1984). Conceptual hydrolic model of flow in the unsaturated zone, Yucca Mountain, Nevade. USGS Water-Resources
-11- Investigations Report 84-4345. Lakewood, CO. 2. West, K. (1988). NNWSI Milestone report, In prepexation. 3. Buddemeir, R.W., and Hunt, J.R. (1987). Radionuclides in Nevada Test Site of groundwater: Transport by clay colloids. Presented before the Division Environmental Chemistry, American Chemical Society, Denver, Colorado. at 4. Brown, C.M., Ellwood, D.C. and Hunter, J.R. (1977). Growth of bacteria 1, surfaces: Influence of nutrient limitations. FEMS Microbiol. Lett. 163-166. 5. Jonsson, P., and Wadstrom, T. (1983). High surface hydrophobicity of Staphylococcus aureus as revealed by hydrophobic interaction chromatography. Curr. Microbiol. 8, 347-353. of 6. Pringle, J.H., Fletcher, M., and Ellwood, D.C. (1983). Selection attachment mutants during the continuous culture of Pseudomonas fluorescens and relationship between attachment ability and surface composition. J. Gen. Microbiol. 129, 2557-2569. 7. Hermansson, M., Kjalleberg, S., Korhonen, T.K., and Stenstrom, T.A. (1982). Hydrophobic and electrostatic characterization of surface structures of bacteria and its relationship to adhesion at an air-water surface. Arch. Microbiol. 131, 308-312. of 8. Doyle, R.J., Nesbitt, W.E., and Taylor, K.C. (1982). On the mechanisms adherence of Streptococcus sanguis to hydroxlapatite. FEMS Microbiol. Lett. 15, 1-5. 9. Lahav, N. (1962). Adsorption of sodium bentonite particles on Bacillus subtilis. Plant and Soil 17, 191-208. 10. Marshall, K.C. (1968). Interaction between colloidal montmorillonite and cells of Rhizobium species with different ionogenic surfaces. Biochem. Biophys. Acta. 156, 179-186. 11. Marshall, K.C. (1969). Studies by microelectrophoretic and microscopic J. techniques of the sorption of illite and montmorillonite to Rhizobia. Gen. Microbiol. 56, 301-306. 12. Marshall, K.C. (1969). Orientation of clay particles sorbed on bacteria possessing different ionogenic surfaces. Biochem. Biophys. Acta 193, 472-474. J. 13. Greenland, D.J. (1956). The adsorption of sugar by montmorillonite. Soil Sci. 7, 319-328. 14. Dianowa, E.W., and Weroschilowa, A.A. (1925). Die adsorption der bakterien
-12- durch den boden (in Russian with a German sunmary). Nauchnoagron. Zh. 2, 520-542. 15. Peele, T.C. (1936). Adsorption of bacteria by soils. Cornell Univ. Agr. Expr. Stat., Memoir 197. 16. Stotzky, G. (1966). Influence of clay minerals on microorganisms. II. Effect of various clay species, homoionic clays, and other particles on bacteria. Cand. J. Miocrobiol. 12, 831-848. 17. Stotzky, G. (1966). Influence of clay minerals on Microorganisms. III. Effect of particle size, cation exchange capacity, and surface area on bacteria. Canad. J. Microbiol. 12, 1235-1246. 18. Stotzky, G., and Rem, L.T. (1966). Influence of clay minerals on microorganisms. I. Montmorillonite and kaolinite on bacteria. Canad. J. Microbiol. 12, 547-563. 19. Stotzky, G., and Rem, LT. (1960). Influence of clay minerals on microorganisms. IV. Montmorillonite and kaolinite on fungi. Canad. J. Microbiol. 13, 1535-1550. 20. Novakova, J. (1972). Effect of increasing concentrations of clay on the decomposition of glucose. I. Effect of bentonite. Zentralb. Bakteriol., Abst. II, 127, 359-366. 21. Novakova, J. (1972) Effect of increasing concentrations of clay on the decomposition of glucose. II. Effect of kaolinite. Zentralb. Bakteriol., Abst. II, 127, 367-372. 22. Filip, Z. (1973). Clay minerals as factors influencing biochemical activity of soil microorganisms. Folia Microbiol. 18, 56-74. 23. Olness, A, and Clapp, C.E. (1972). Microbial degradation of a montomorillonite-dextran complex. Soil Sci. Soc. Amer. Proc. 36, 179-181. 24. Kunc, F., and Stotzky, G. (1970). Breakdown of some aldehydes in soils with different amounts of montmorillonite and kaolinite. Folia Microbiol. 15, 216. 25. Weber, J.B., and Coble, H.D. (1968). Microbial decomposition of diquot adsorbed on montmorillonite and kaolinite clays. J. Agr. Food Chem. 15, 475-478. 26. Hance, R.J. (1974). Soil organic matter and the adsorption and decomposition of the herbicides atrazine and linuron. Soil Biol. Biochem. 6, 39-42. 27. Harter, R.D., and Stotzky, G. (1973). X-ray diffrac4ton, electron
-13- microscopy, electrophoretic mobility, and pH of some stable smectite protein complexes. Soil Sci. Soc. Amer. Proc. 37, 116-123. 28. McLaren, A.D. (1957). Concerning the pH dependence of enzyme reduction on cells, particulates and in solution. Science 125, 697. 29. Goren, M.B. (1968). Process for flocculating finely divided solids suspended in an aqueous medium with a microbial polysaccharide. U.S. Patent No. 3,406,114. 30. Bomstein, R.A. (1972). Waste treatment with microbial nucleoprotein flocculating agent. U.S. Patent No. 3,684,706. 31. Brown, M.J., and Lester, J.N. (1979). Metal removal in activated sludge: The role of extra-cellular polymers. Water Research 13, 817-837. 32. Obayashi, A.W., and Gaudy, A.F. (1973). Aerobic digestion of extracellular microbial polysaccharides. J. Water Poll. Contr. Fed. 45, 1584-1594.
-14- Figure 1. Number of individual clay particles per 5.0 x 10-' ml in the presence of a Pseudomonas sp. 25
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